JOURNAL OF EXPERIMENTAL ZOOLOGY 301A:150–159 (2004)
Forelimb Spike Regeneration in Xenopus laevis:
Testing for Adaptiveness
ROY A. TASSAVA
Department of Molecular Genetics, The Ohio State University, Columbus,
Ohio 43210
ABSTRACT
Experiments were designed to test adaptability of forelimb spike regenerates in
Xenopus laevis froglets. The results show that when amputation is at the radius/ulna level,
regeneration occurs in 100% of the cases and a single spike of cartilage is the result. The spike
regenerates originating from radius/ulna level amputations can be used for feeding and froglet
growth is only minimally compromised by the spike. The spike grows in length as the froglet body
grows and thus is in homeostasis with the body. The spike develops nuptial pad tissue in
reproductively mature males and is occasionally molted, indicating responsiveness to gonadal and
thyroid hormones. Finally, and most important, the spike can be used for amplexus and successful
mating. In contrast, spikes originating from humerus level amputations were considerably shorter
and regeneration from that limb level was less frequent. When amputation was at the body wall
regeneration did not occur. J. Exp. Zool. 301A:150–159, 2004. r 2004 Wiley-Liss, Inc.
INTRODUCTION
The South African Clawed frog, Xenopus laevis,
uses its forelimbs mainly for feeding and amplexus. The forelimbs are shaped and anatomically
positioned so that immediately upon metamorphosis and then throughout life the frog can use
them to ‘‘scoop’’ food into its mouth. Thus the
autopod is perpendicular to the substrate rather
than flat as seen with most anurans. The male frog
is about two-thirds the size of the female but
nevertheless can grasp the female firmly just
anterior to the hind limbs around the base of the
abdomen during amplexus. It thus is positioned
ideally to deposit sperm on the newly released
oocytes. This is accomplished in part by the
geometry of the forelimb and also by the irregular
and highly cornified epidermis (‘‘black sticky
hairs,’’ Deuchar, ’75; ‘‘nuptial excrescences,’’
Duellman and Trueb, ’86) that develop exclusively
on the inner surfaces of the forelimb autopod and
zeugopod (digits, palms, and forearms) in the
sexually mature male. Even though the female
skin is covered by mucus and is very slippery, the
male holds the amplexus position until mating is
completed. While swimming, the frog is propelled
almost exclusively by the hindlegs; the autopod is
again geometrically positioned to get the most
forward thrust from the push backwards. The
hindlimb digits also contain keratinized ‘‘claws’’
which are often used to tear food into small pieces
r 2004 WILEY-LISS, INC.
which the forelimbs then ‘‘push’’ into the mouth
(Deuchar, ’75). Thus, both fore- and hindlimbs are
arguably highly adaptive for Xenopus in its aquatic
environment.
Xenopus, as is true for most anuran amphibians,
can regenerate complete fore- and hindlimb
patterns during early stages of metamorphosis
but in later stages the pattern of the regenerated
structure becomes more and more hypomorphic
(Dent, ’62).
Nevertheless, froglets and adult frogs retain the
ability to regenerate a muscle-deficient, cartilagenous spike. Based on histological analysis,
Korneluk and Liversage (’84a) suggested that
spike regeneration in Xenopus represents a
‘‘dominant tissue regeneration response’’ in contrast to epimorphic regeneration seen in newts.
Epimorphic regeneration involves release of differentiated cells from their tissues and acquisition
of pluripotency in developmental potential, i.e.
dedifferentiation, and their proliferation and
accumulation to form a blastema. Tissue regeneration, in contrast, involves the proliferation of
cells with limited developmental potential, such as
fibroblasts, without dedifferentiation but with
some limited tissue replacement (reviewed in
n
Correspondence to: Roy A. Tassava, Department of Molecular
Genetics, 484 W. 12th Ave., Columbus, OH 43210.
E-mail: [email protected]
Received 22 April 2003; Accepted 1 October 2003
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/jez.a.20015
XENOPUS REGENERATION IS ADAPTIVE
Korneluk and Liversage, ’84a). That the Xenopus
spike regenerate represents epimorphic regeneration has been argued based on both grafting (Goss
and Holt, ’92) and molecular (Endo et al., 2000)
data.
Whether or not limb regeneration is evolutionarily adaptive or inherent has been discussed by
Goss (’92) and Thouveny and Tassava (‘98). Goss
(’92) states, ‘‘If regeneration were adaptive, it
would have arisen autonomously by natural
selection from non-regenerative antecedents. Unless each episode coincidently reinvented the same
method of regeneration independently, one would
expect the various lineages to differ basically from
each other, which they do not. On the other hand,
if regeneration were inherent to metazoan life, a
derivative of embryogenesis, its various expressions should be as much like each other as they
resemble the development of embryonic appendage buds, which they do. It follows that the
uneven distribution of regeneration must have
been due to its extinction here and there, not as a
negative adaptation by natural selection but
as a pleiotropic epiphenomenon linked to more
useful adaptations with which it became incompatible.’’ Goss (’92) goes on to argue that these
latter adaptations included the transition from
water to land habitats and the physiological
change from poikilothermy to homeothermy.
Later he states, ‘‘Only by being subjected frequently enough to the acid test of natural selection
can a structure or process be preserved in the
course of evolution.’’ Goss (’92) favored the
‘‘inherent’’ idea, at least as regards vertebrate
limb regeneration, a process restricted largely to
urodele amphibians.
The issue of inherent vs adaptive is discussed by
Thouveny and Tassava (‘98) who point out that
one interpretation of the adaptive view regarding
epimorphic regeneration would be that early
vertebrates initially did not have the capacity to
regenerate and that the process evolved in some
species by natural selection. They further suggest
that one selective pressure for urodele limb
regeneration ability might have been sibling
chewing due to decreasing pond size and increasing population density, a phenomenon readily
demonstrated in laboratory populations of urodeles. Furthermore, when urodele larvae (Ambystoma tigrinum) are reared in high density, they
are likely to become cannibalistic (Pfennig and
Collins, ’93). Wagner and Misof (’92) suggest
multiple independent origins of impaired regeneration in teleosts and amphibians.
151
The present investigation was designed to test
the hypothesis that the pattern deficient spike
regenerates in Xenopus are adaptive. Measurements were done to assess whether froglets with
one or both forelimb spikes could eat adequately
and grow normally to adult stages. Measurements
assessed whether the initial regenerated spikes
subsequently grew as the frog grew and thus could
be said to be in homeostasis with the body. Related
to the test of adaptiveness, the extent of regeneration was compared at the stylopodium vs zeugopodium vs body wall levels. Finally, tests were
carried out to assess whether frogs that had grown
and matured sexually with one or two forelimb
spikes could undergo amplexus and reproduce.
MATERIALS AND METHODS
Xenopus froglets and adults were raised from
tadpoles fed strained baby food (peas and beans).
Hindlimbs were present by 15 days after hatching
(stage 48; Nieuwkoop and Faber, ‘56) and the first
froglets metamorphosed by seven weeks (see also
Brown, ’70). Newly metamorphosed froglets were
randomized by size and housed at a density
of 10–12 froglets per clear, plastic container in
2 liters of water at a depth of 1.5 inches and were
fed small pieces of beef liver once daily. Liver was
introduced at a rate of 4–5 pieces per froglet per
day but some froglets showed greater assertiveness and consumed 6–7 pieces/day. Water was
changed daily.
Within one or two days of completion of metamorphosis, assessed by the complete loss of the tail
(stage 66), froglets were lightly anesthetized in
0.005 % MS 222 (methane sulfonate; Sigma) and
forelimbs were amputated either through the
radius/ulna (R/U; zeugopodium), humerus (H;
stylopodium), or at the body wall. Amputations
were randomly made at the R/U and H levels such
that the plane of amputation varied from more
distal, to middle, to more proximal levels of the
respective skeletal elements. In each of two
containers with 12 frogs each, two frogs had both
forelimbs amputated at the R/U level, six frogs had
only one forelimb amputated at the R/U level, and
four frogs served as unamputated controls. In a
third container of 12 frogs, four frogs had both
forelimbs amputated at the H level, four frogs had
one forelimb amputated at the mid-H level, and
four frogs served as unamputated controls. In a
fourth container of 12 frogs, four frogs had one
forelimb amputated at the body wall, four frogs
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R. A. TASSAVA
had both forelimbs amputated at the body wall,
and four frogs served as unamputated controls.
It was not possible to determine the sex of the
frogs until reproductive maturity, when the forelimbs of the males developed nuptial pad tissue
and the size difference between males and females
became evident.
The frequency and extent of regeneration were
assessed through time at the three amputation
levels. Frog body growth was assessed and
compared between the unamputated, single forelimb amputated at the R/U level, and dual
forelimbs amputated at the R/U level. The mean
of the body lengths of the control, unamputated
frogs was determined and the body lengths of the
two experimental groups were expressed as the
percent of the mean of the control, unamputated
frog body lengths. Length of the regenerate was
determined by measuring from the mid-elbow to
the tip of the regenerate and thus included a short
portion of the stump. The mid-elbow was utilized
as a baseline point because the amputation level
became less clear as regeneration progressed.
Growth of the regenerates was examined in
proportion to the growth of the frog, means were
determined, and the data were expressed as
percent of the control limb length from the midelbow to the tip of the longest digit.
A total of seven unamputated frogs (four males
and three females) and 10 frogs with a single spike
regenerating at the R/U level (four males and six
females) were raised to adulthood. When the frogs
reached reproductive maturity, at 18–24 months
of age (see Brown, ’70; Deuchar, ’75), three
control, unamputated males and three males with
one forelimb spike were paired with either
unamputated females or single-spike females.
The appropriate human chorionic gonadotropic
hormones (Sigma) were administered by injection
into the dorsal lymph sac (Brown, ’70; Sive et al.,
2000) and observations were made of the occurrence or absence of amplexus and whether fertile
eggs were obtained, as assessed by observations of
embryonic development.
After observations on mating, some limbs were
sampled for histological analysis. Limbs were fixed
in Bouin’s fixative, dehydrated and cleared, embedded in paraffin, sectioned serially, and deparaffinized sections were stained with hematoxylin
and eosin. Unamputated forelimbs and single
spike regenerates were examined in males to
evaluate the presence and extent of nuptial pad
tissue. Spike regenerates were sectioned longitudinally to evaluate the melding of the regener-
ate cartilage to the stump bones. Also, a single
spike regenerate that was ‘‘floppy’’ and not
melded to the stump bones was sectioned longitudinally and examined by histology.
RESULTS
Blastemas were present by 10 days after
amputation at which time it could be ascertained
whether a limb would or would not regenerate.
Every limb regenerated when amputation was at
the R/U level; single spike regenerates were
invariably produced, sometimes with slight bends
(Fig. 1a). In one case a distally bifurcated
regenerate was seen (not shown). A lower proportion of froglets regenerated when amputation was
through the H (8 of 10 limbs regenerated) and the
spikes were considerably shorter than those at the
R/U level (Fig. 1b). In one case an elbow-like bend
was seen (Fig. 1b, 4th frog from the left). Of the 12
forelimbs amputated at the body wall, none
regenerated (Fig. 1c).
Froglets completed forelimb spike regeneration
by five weeks after amputation, far before body
growth was completed. The spike regenerate
then grew in relatively good proportion to the
body. At one month after amputation, the lower
arm (zeugopodium stump plus regenerated spike)
represented on average 38% of the length of the
body. At the same age, with unamputated froglets,
the length of the zeugopodium plus the autopodium represented on average 40% of the body
length. Also, the average length of the zeugopodium stump plus the regenerated spike in proportion to that of the zeugopodium plus autopodium
of intact forelimbs was 98%. At 10 months, when
froglets had nearly reached adult size, spike
regenerates were again measured in proportion
to the opposite unamputated forelimb. The proportion on average was now only somewhat less,
being 90%. The above results are based on average
spike and body lengths of at least five froglets
per measurement. It can be seen in Fig. 1a that
the forelimbs with the spikes regenerating from
the R/U level are very nearly equal in length to the
contralateral, unamputated forelimbs.
Variation in lengths of the regenerates was
possibly related to inexact levels of amputation
but an insufficient number of cases was available
to carefully examine this possibility. Nevertheless,
forelimbs amputated through the proximal portion of the R/U seemed to regenerate shorter
spikes than forelimbs amputated through the
distal R/U. For the proportional comparisons
XENOPUS REGENERATION IS ADAPTIVE
Fig. 1. Photos of froglets 5 weeks after amputation
through the radius/ulna (1a), the humerus (1b) and at the
body wall (1c). Note in 1a that the spike regenerates are
nearly the same length as the opposite, unamputated
forelimb. Arrow points to the amputation level of the froglet
at the left in the figure. In 1b it can be seen that the spike
regenerates are much shorter when amputation is through
the humerus. The arrow indicates the level of amputation of
153
the frog at the left in the figure. The frogs in the figure
represent 4 of the 8 limbs that regenerated (out of a total of
10) when amputation was at the humerus level. The 4 frogs
shown in Fig. 1c are typical of the non-regeneration seen
when amputation is at the body wall (see arrow pointing to
level of amputation of 2nd frog from the left). Bar in 1a
(equivalent in 1b, 1c)¼10 mm. Photos represent actual size of
frogs.
154
R. A. TASSAVA
below, froglet growth was assessed by determining
the means of the body lengths for 4–5 randomly
chosen froglets previously amputated at the R/U
level. Froglets with two intact forelimbs showed
the most rapid body growth with mean body
weights increasing from 0.55 g on day 0 (day of
amputation) to 0.954 g at one month after
amputation, to 10.2 g at four months after
amputation. Froglets with one leg amputated
increased from 0.54 g at day 0 to 0.90 g at one
month and 9.4 g at four months. Froglets with
both forelimbs amputated showed mean body
weights increased from 0.53 g at day 0 to 0.750 g
at one month to 8.8 g at four months. Thus,
froglets with both forelimbs intact exhibited the
most growth; froglets with both forelimbs amputated exhibited the least growth; and froglets with
only one forelimb amputated exhibited intermediate growth. Expressed as percent of control,
froglet mean body weight was less at one month
for the froglets with both limbs amputated (79% of
control) than at four months (86% of control)
whereas for the singly amputated frogs, the
percent of control was 94% at one month and
92% at four months. When forelimb regeneration
was completed and spike regenerates reached
maximum lengths, froglets could utilize the spikes
for feeding, and body growth rate was increased
Fig. 2. Ventral view of a male frog exhibiting breeding
plumage. Nuptial pad tissue is apparent on the inside of the
left, unamputated limb and inside of the right, amputated
limb, extending from the stylopodium (arrow) to the zeugo-
but was still not equivalent to that of froglets with
both forelimbs intact. Frog feeding was hindered
more by having two spikes than by having one
spike.
The single-spike frogs that were raised to
adulthood (seven months and beyond) grew to
approximately the same size and weight as the
unamputated frogs. Average weights were not
determined because the sex of individual frogs
began to influence the weight and size, with the
weights of females ultimately ranging from 85–125
grams and the weights of males ranging from
45–55 grams (Brown, ’70).
Growth data were not collected on froglets with
limbs amputated at the body level but it became
obvious early on that these froglets struggled to
obtain food, particularly if both forelimbs had
been amputated. Froglets amputated at the body
wall were not raised to adulthood.
Upon reaching sexual maturity, Xenopus males
developed typical nuptial pad tissue on the inside
surface of the autopodium and zeugopodium
(Fig. 2) and this nuptial pad tissue became more
pronounced after the hormonal injections (not
shown; see Brown, ’70). None of the female frogs
developed nuptial pad tissue (see Fig. 6). The
spikes that developed from R/U level amputations
also developed nuptial pad tissue, whether one or
podium and autopodium of the unamputated limb and to the
tip of the regenerated spike. The black claws at the tips of the
hindlimb toes can be seen. Bar¼1 cm.
XENOPUS REGENERATION IS ADAPTIVE
both forelimbs were amputated, as seen with the
unaided eye (Figs. 2, 3, and 5), the dissecting
microscope, and by histology (Fig. 4a,b). The
epidermis and the nuptial pad tissue was occasionally molted (Fig. 4a). Amplexus behavior in
males is reflexive and a male will clasp onto a
researcher’s finger (Deuchar, ’75). The ‘‘stickiness’’ of the intact forelimb nuptial pad vs. the
nuptial pad tissue on the spikes seemed equivalent, as tested with a finger by two independent
testers. Also, the density of the black, nuptial
excrescences appeared similar between unamputated forelimbs and regenerated spikes but quantitation was not attempted. Each of the black
nuptial excrescences appeared to be associated
with a specialized group of epidermal cells immediately below it (Fig. 4b). All three male frogs
with intact forelimbs exhibited amplexus behavior. Amplexus is lumbar in Xenopus as opposed to
axial as in Rana (Brown, ’70). All three of the
females produced fertile eggs with percent fertilities of 78%, 75%, and 71%. Likewise, all three
male frogs with one intact and one forelimb spike
that had regenerated from the mid-radius/ulna
level exhibited attempts at amplexus behavior but
only two of the frogs were able to amplex
successfully (Fig. 6) and mate. These two matings
resulted in eggs with percent fertilities of 74% and
72%. It was estimated that in each of the latter two
155
cases the female released over 200 eggs. The single
spike frog that did not successfully amplex was
subsequently noted to have a ‘‘floppy’’ spike that
was not melded to the cut bones. Analysis of serial
sections of this forelimb after H&E staining
confirmed this observation (not shown). The
cartilagenous spike regenerate in Xenopus is
normally continuous with and firmly melded into
the bones of the stump, as seen by histology (not
shown; see Korneluk and Liversage, ’84a).
The frogs amputated at the H level were not
raised to adulthood. It seems likely that these
spikes would have been too short to enable the
frogs to undergo amplexus with the respective
female, even if one forelimb was intact. It is not
known if the short spikes regenerated from the
mid-humerus level would have developed nuptial
pad tissue.
DISCUSSION
The results are consistent with the view that
Xenopus forelimb spike regenerates originating
from amputations at the R/U level are adaptive.
First, froglets were able to utilize the spikes for
feeding under laboratory conditions. Second, once
regenerated, the spikes grew proportionally to
the growth of the body, suggesting that they were
in homeostasis with the body. Third, in males,
Fig. 3. Ventral view of a male frog with regenerated spikes on both forelimbs. Both spikes developed nuptial pad tissue.
Bar¼1 cm.
156
R. A. TASSAVA
Fig. 4. Micrographs of histological sections showing the
black, horn-like nuptial excrescences on the inner portion of
the regenerated spikes in male frogs. In 4a it can be seen that
the outer epidermal layer of the nuptial pad tissue is
occasionally molted (arrow). g¼gland. c¼cartilage of the spike
extending across the figure. Distal is to the right in both 4a
and 4b. bar¼0.3 mm. 4b is a higher magnification showing the
black, horn-like nuptial excrescences angled to maximize
grasping of the female. Note that each excrescence seems to
originate from a specialized group of epidermal cells immediately below it (arrow). g¼gland. bar¼0.1 mm.
nuptial pad tissue developed along the inner limb
surface which was occasionally molted, thus
indicating that the skin was responsive to the
relevant testicular and thyroid hormones (Duellman and Trueb, ’86). If any of the above results
were not seen, one could argue that the spike
regenerates were not adaptive. However, these
observations alone would not indicate adaptiveness if frogs with one or more forelimb spike
regenerates were unable to breed. Thus, the
fourth observation, that male frogs were able to
utilize the spikes in amplexus and breeding, taken
together with observations 1–3, lends strong
support to the hypothesis that forelimb spike
regeneration in Xenopus is adaptive.
The adaptiveness of Xenopus regenerates is
consistent with the views of Goss and Holt (’92)
and Endo et al. (2000) that this spike comes about
by epimorphic regeneration. Goss and Holt (’92)
reasoned that the key feature of epimorphic
regeneration is blastema formation and, furthermore, that blastema formation requires a functional wound epithelium (see Mescher, ‘76). Thus
wound epithelium formation was prevented by
inserting freshly amputated limbs into the body
cavity. Such limbs did not form a blastema,
leading Goss and Holt (’92) to conclude that
Xenopus spike regeneration is epimorphic. Endo
et al. (2000) utilized probes for molecular markers
of urodele blastema formation and observed the
same markers in Xenopus blastemas, again concluding that Xenopus regeneration is epimorphic.
It remains to be determined what patterning
genes are not expressed during Xenopus regeneration and why selection was not rigorous enough to
result in anterior/posterior regenerate patterning
in this frog.
It is of interest that when amputations occurred
at the H level, regeneration either did not occur or
was very poor. The spikes that did regenerate
were less than half the length of the spikes
regenerating at the R/U level. Whether or not H
level spikes would have developed nuptial pad
tissue upon sexual maturity was not determined.
Finally, it seems likely that frogs with the short H
level spikes, even with nuptial pad tissue, would be
unable to amplex with female frogs. If a spike
regenerated from the H level, even if it was the
length of the missing limb portion, amplexus
would not be possible without an elbow joint or
without a bend in the medial direction. It follows
that there would be no adaptive value to regenerate from the humerus level, consistent with the
present results. Certainly spikes emanating from
the body wall, if regeneration occurred from that
level, could not be used in amplexus and would not
be adaptive. Predictably, as shown here, regeneration does not occur from the body wall level (see
also Gallien and Beetschen, ’51). It should be
noted that adult Xenopus also regenerate better
from distal levels and there is no regeneration
from very proximal levels (Gallien and Beetschen,
’51), as seen here with froglets.
The spike originating at the R/U level is tightly
melded into the cut ends of the bones, as noted
previously (Korneluk and Liversage, ’84a). In the
single case here, wherein the spike was not melded
XENOPUS REGENERATION IS ADAPTIVE
157
Fig. 5. A dorsal view of a male frog showing nuptial pad
tissue visible on the autopod of the unamputated left forelimb
and on the spike regenerate of the right forelimb. This frog is
shown in amplexus with a female in Fig. 6. bar¼1 cm.
Fig. 6. A dorsal view of the male frog shown in Fig. 5 in
amplexus with a female. The photograph is somewhat reduced
in size compared to Fig. 5. Note that the right forelimb with
the spike regenerate has a good grasp of the female. This pair
of frogs had a successful mating. The female has a spike
regenerate on the left but neither forelimb exhibits nuptial
pad tissue. Note the larger size of the female. Bar¼1.5 cm.
into the stump bones, amplexus was not possible.
In this regard, it should be noted that spike
regeneration occurs even if the stump bones are
not present at the level of amputation (Korneluk
and Liversage, ‘84b) but such spikes probably
could not be used in amplexus. Some spikes
158
R. A. TASSAVA
(see Figs. 1 and 2) showed bending to various
degrees, indicative of joint formation. However, in
a preliminary histological study of Xenopus
froglet regenerates that showed bending, only
one of six had what appeared to be a joint; the
other five were composed of solid cartilage
(Tassava, unpublished). If a froglet regenerate
did have a joint, then the musculature necessary
for maintaining the distal segment in the amplexus position would necessarily have to
be regenerated. But, Xenopus regenerates are
noted for their absence of muscle (Korneluk and
Liversage, ‘84a).
If forelimb spike regeneration is adaptive, the
question arises as to what factor or factors
caused limb loss (and thus adaptive pressure for
regeneration) in evolutionary history. Predation
is perhaps the most likely. Limbs of late tadpoles
and young froglets are somewhat fragile,
certainly at least as fragile as limbs of Ambystoma
larvae. The latter are often chewed off to varying
degrees by siblings. However, Xenopus froglets
do not chew legs of siblings in the laboratory,
at least under conditions of ad libitum feeding, as
in the present experiment. It would be of interest
to examine natural populations of Xenopus to
determine if froglet limbs are chewed by siblings
or by predators. It is possible that froglet limbs
would be chewed by adult frogs if sharing the same
pond or even that larger froglets might chew the
limbs of smaller froglets if in dense conditions.
Note that adults will eat tadpoles (Deuchar, ’75).
Finally, it might seem to be intuitively obvious
that hind limbs would be lost more often than
forelimbs. Hindlimbs are present for a good part of
tadpole life, i.e. during all of pro-metamorphosis,
and are long and perhaps more available to be
grasped by predators. To be adaptive, the hindlimb regenerative spike would have to be useful
for swimming, either to find food or escape
predation.
The present results are interesting with
regard to the view favored by Goss (‘92) that
limb regeneration is inherent, as opposed to
being adaptive. In attempting to apply this view
to Xenopus spike regeneration, it would seem
that initially this frog species would have been
capable of regenerating a complete pattern and
then for some reason the ability to regenerate
a pattern was lost. The two main reasons for loss
of inherent regenerative ability in non-regenerating vertebrates are acquisition of a terrestrial
habitat and warm-bloodedness (Goss, ‘92). Since
Xenopus is poikilothermic and aquatic, there
seems be no intuitive reason as to why this species
should not have retained the ability to regenerate
a complete pattern. On the other hand, as noted
above and a view favored by the present results,
selective pressure may have been sufficient to
result in Xenopus spike regeneration but with
selection pressure not strong enough for pattern
regeneration.
Alternatively, the rigorous foreleg and
hindleg movements in Xenopus possibly precluded
the regeneration of a pattern due to the fragile
nature of the intermediate blastema stages.
Xenopus is the most primitive of the three
genera in the family Pipidae. It is difficult to
hypothesize about gain or loss of regeneration in
Xenopus without knowing if this species was
terrestrial at one time and then returned to water,
or never evolved the ability to live on land (see
Deuchar, ’75).
The initial regenerates of the froglets did
not show dorsal/ventral or anterior/posterior
patterning but when the male froglets matured,
nuptial pad tissue always formed on the ventral
surface, indicative of a dorsal/ventral pattern.
Whether this pattern is the result of patterning mechanisms or merely due to contributions
of pad-potential cells from the stump skin to
the regenerate remains to be determined. In
attempts to stimulate pattern regeneration
in Xenopus, factors could be utilized that are
known to be involved in urodele regeneration,
wherein a complete pattern is the norm. One
such factor, retinoic acid, resulted in cartilage
condensations with ‘‘complex cartilage distal
subdivisions’’ (Crawford and Liversage, ’92) or
had very little effect (Tassava, unpublished) when
applied to Xenopus limb stumps. From the results
of Endo et al. (2000) it would seem logical to
express a sonic hedgehog transgene alone or in
combination with dorsal-ventral patterning genes
in the Xenopus limb stump. Another approach
would be to ‘‘remove presumed inhibitions’’ (Goss,
’80). Finally, the developmental program necessary to regenerate a spike is itself likely to be
complex and some effort would be necessary to
elucidate all of its complexities in order to modify
it, in favor of a patterning program (Thouveny and
Tassava, ‘98).
ACKNOWLEDGEMENTS
The author is grateful to Anthony L. Mescher
for many helpful suggestions on the manuscript.
XENOPUS REGENERATION IS ADAPTIVE
LITERATURE CITED
Brown AL. 1970. The African clawed toad, Xenopus laevis. A
guide for Laboratory Practical Work. London, UK: Butterworth and Company. p 1–124.
Crawford M, Liversage RA. 1992. Pattern perturbation in
Xenopus laevis forelimb regenerates following treatment
with retinoic acid and carrier media. In: Taban CH, Boilly B,
editors. Keys for regeneration. Monogr Dev Biol Basel,
Switzerland: Karger. p 146–156.
Dent JN. 1962. Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad. J
Morph 110:61–77.
Deuchar EM. 1975. Xenopus: the South African clawed frog.
New York: John Wiley & Sons. P 1–71.
Duellman WE, Trueb L. 1986. Biology of the amphibians.
Baltimore, Maryland: Johns Hopkins Press. p 51–86.
Endo T, Tamura K, Ide H. 2000. Analysis of gene expressions
during Xenopus forelimb regeneration. Dev Biol 220:296–
306.
Gallien L, Beetschen J. 1951. Extension et limites du pouvoir
regenerateur des membres chez Xenopus laevis. C R Soc Biol
145:874–876.
Goss RJ. 1980. Prospects for regeneration in man. Clin Orthop
151:270–282.
Goss RJ, Holt R. 1992. Epimorphic vs tissue regeneration in
Xenopus forelimbs. J Exp Zool 261:451–457.
159
Goss RJ. 1992. The evolution of regeneration: adaptive or
inherent? J Theor Biol 159:241–260.
Korneluk RG, Liversage RA. 1984a. Tissue regeneration in the
amputated forelimb of Xenopus laevis froglets. Can J Zool
62:2383–2391.
Korneluk RG, Liversage RA. 1984b. Effects of radius-ulna
removal on forelimb regeneration in Xenopus laevis froglets.
J Emb Exp Morph 82:9–24.
Mescher AL. 1976. Effects on adult newt limb regeneration of
partial and complete skin flaps over the amputation surface.
J Exp Zool 195:117–127.
Nieuwkoop PD, Faber J. 1956. Normal table of Xenopus laevis
(Daudin). North-Holland, Amsterdam. p 1–64.
Pfennig DW, Collins JP. 1993. Kinship affects morphogenesis
in cannibalistic salamanders. Nature 362:836–838.
Sive HL, Grainger RM, Harland RM. 2000. Early development of Xenopus laevis: A laboratory manual. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
p 100.
Thouveny Y, Tassava R. 1998. Regeneration through phylogenesis. In: Ferretti P, Geraudie J, editors. Cellular and
molecular basis for regeneration. New York: John Wiley &
Sons. p 9–43.
Wagner GP, Misof BY. 1992. Evolutionary modification
of regenerative ability in vertebrates: a comparative
study on teleost pectoral fin regeneration. J Exp Zool
261:62–78.