/ . Embryol. exp. Morph. Vol. 63, pp. 243-265, 1981
Printed in Great Britain © Company of Biologists Limited 1981
243
Experiments on Anuran limb buds and their
significance for principles of vertebrate
limb development
By M. MADEN 1
From the Developmental Biology Division, National Institute for
Medical Research, London
SUMMARY
A standard set of six experiments performed on the limb buds of two species of Anurans Rana temporaria and Xenopus laevis -are described. The experiments are limb-bud amputation, distal to proximal shifts, proximal to distal shifts, inversion of the dorsoventral axis
inversion of the anteroposterior axis and inversion of both axes. The results are compared to
those previously reported for Urodeles and chicks to determine whether any principles of
vertebrate limb development can be formulated. It appears that the proximodistal axis
becomes increasingly mosaic from the Urodeles through Anurans to chicks. In the transverse
axes however, there is much more uniformity of behaviour in the production of supernumerary limbs. The relation between the type of limb development (regulative or mosaic)
and the subsequent regenerative powers of the adult limb is discussed.
INTRODUCTION
To formulate universal laws of development we need to compare the results of
experiments on many organisms throughout the animal kingdom. As far as
vertebrate limb development is concerned we cannot yet make such generalisations largely because of the way in which this subject has been investigated. It is
usual to study in great detail the most readily available laboratory organism of
the day rather than to study in lesser detail a wider range of animals. Thus, work
performed in the early part of this century mostly employed Urodeles, particularly Ambystoma species (Harrison, 1921; Swett, 1927), whereas more recently
the chick limb bud has become the most studied system (see for example Ede,
Hinchliffe & Balls, 1977). This situation is particularly unfortunate since the
development of these two types of limb seems to be quite different - the Urodele
limb is highly regulative (Harrison, 1918; Slack, 1980; Maden & Goodwin,
1980), whereas the chick limb is much more mosaic (Warren, 1934; Summerbell
& Lewis, 1975; Summerbell, 1977).
1
Author's address: Developmental Biology Division, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
244
M. MADEN
Furthermore, because different experimenters have asked different questions
of developing limbs there has not been a consistent set of experiments which has
been performed on the limb buds of these animals. A previous publication
(Maden & Goodwin, 1980) began an attempt to provide such standardisation by
reporting the results of a set of operations on Ambystoma mexicanum limb buds.
The data for the same experiments on chick limb buds can be extracted from the
literature for comparison (see Discussion). In the present paper, the results of
that same set of experiments performed on the limb buds of two species of
Anurans, Rana temporaria and Xenopus laevis are described. Apart from permitting more valid generalisations to be made about vertebrate limb development, this work is also relevant to the question of whether the mechanisms of
limb development dictate the subsequent regenerative capabilities of the limb. In
other words, is the adult chick limb (and thus by inference the adult human limb)
incapable of regeneration because of the mosaic nature of its development and,
conversely, can the adult Urodele limb regenerate because of the regulative
nature of its development?
MATERIALS AND METHODS
The experiments were performed on Rana temporaria obtained as spawn from
local ponds and reared in the laboratory and Xenopus laevis bred from laboratory
stock. The hind limb buds were used since these are much more accessible than
forelimbs in Anurans. Larvae were anaesthetised in 1 :3000 MS222. Two stages
of limb buds were used, those of Rana temporaria (Fig. 1 a and b) being equivalent to limb bud stages IV and V of Rana pipiens (Taylor & Kollros, 1946 - no
staging of Rana temporaria to include the limb buds could be found) and for
Xenopus stages 51 and 52 of Nieuwkoop & Faber (1967) (Fig. 1 c and d). Most
operations, that is limb-bud removal (Series 1) and all the rotations (Series IVVII), were performed at the earlier of the two stages. The distal to proximal
grafts (Series II) were performed at the later of the two stages and for proximal
to distal grafts (Series III) whole limb buds of the earlier stages were grafted to
distal levels of later-stage buds. Thus the methodology was intentionally identical to previous work on Ambystoma mexicanum (Maden & Goodwin, 1980) for
comparative purposes.
The operations were performed in air and the correct orientation of limb buds
was easily verified in these two species thanks to the fortuitous presence of a patch
of melanophores on the dorsal surface of the bud (Fig. 1). After transplantation
the animals were placed at 4 °C for 5-10 min to facilitate sticking of the graft
(usually a clot of blood served as glue), and then returned to water. Those larvae
whose grafts did not survive were discarded the next day. During the subsequent weeks the animals were regularly observed and after 5-8 weeks the limbs
were fixed and stained with Victoria blue to reveal their skeletal structure.
Anuran limb-bud development
245
(c)
Fig. 1. Stages of limb development when the operations were performed. The dots
represent melanophores in the limb buds, (a) Early stage of Rana temporaria equivalent to stage V of Rana pipiens (Taylor & Kollros, 1946) when the rotations (Series
IV-VII) and simple amputations (Series I) were performed by cutting at the level
marked A. (b) Later stage of Rana temporaria equivalent to stage VI of Rana pipiens
when the distal to proximal (Series II) and proximal to distal (Series III) grafts were
performed, (c) Stage-51 limb buds (Nieuwkoop & Faber, 1967) of Xenopus laevis
when the rotations (Series IV-VII) and simple amputations (Series I) were performed
by cutting at the level marked A. (d) Stage-52 limb buds of Xenopus laevis when the
distal to proximal (Series IV) and proximal to distal (Series III) grafts were performed.
Bar = 1 mm.
RESULTS
Series I. Limb-bud removal
Whole left limb buds were removed at level A in Fig. 1 a and c for transplanting to the contralateral side in Series V and VI. The severed left stumps thus
served as experimental material to examine their regulative powers. In Rana, a
total of 38 limbs were stained, 36 of which produced perfect 5-digit limbs
(Table 1, Fig. 2). The remaining two limbs had relatively minor defects in the
form of a shortened and curved tibia and fibula.
This excellent ability of Rana to regulate for limb-bud extirpation is not
paralleled by Xenopus. Here a total of 58 cases were examined, only 22 of which
(38 %) produced perfect limbs (Table 1, Fig. 3). Eleven cases gave nothing more
than a femur which terminated about one half of the way down its length; this
was presumably the level at which the bud was removed. The remaining 25 limbs
were defective in one of two ways. The majority (15) had varying degrees of
246
M. MADEN
Table 1. Summary of the results of three types of operation on the proximodistal
axis of axolotl {data from Maden & Goodwin, 1980), Rana and Xenopus {results
reported here) and chick {data from various sources noted below) limb buds assembled into one table for comparative purposes
Result
Nothing
deletion
Perfect
limb
Repeated
limb
Operation
(%)
(%)
(%)
(V)
Axolotl
Rana
Xenopus*
Chickf
Bud removal
Bud removal
Bud removal
Bud removal
0
0
19
100
0
5
100
95
26
38
0
0
—
—
—
Axolotl
Rana
Xenopus
Chick*
D-*P
D->P
0
0
0
0
0
58
100
100
100
42
0
0
—
—
—
0
0
0
0
0
0
0
0
0
0
0
0
P-D
Animal
Axolotl
Rana
Xenopus
Chick§
D^P
D^P
PVD
P-»D
P-*D
P+D
100
100
100
100
* Not including the half-limbs noted below.
t Data from Lillie (1904), Shorey (1909) Peebles (1911), Spurling (1923), Saunders (1948)
and Hampe (1959).
X Data from Summerbell (1977).
§ Data from Summerbell & Lewis (1975).
proximodistal deletions in the form of shortened or deleted elements (Fig. 4).
The other 10 limbs were classified as half-limbs, that is they were proximodistally complete but only contained one element in the zeugopodium and
carpals and only 2-4 digits (Fig. 5). Nine of these were identified as posterior
halves and one anterior half limb. This strange result could be due to cutting
inaccuracies, that is, amputating the limb bud on a slant rather than at right
Fig. 2. A perfect hind limb of Rana temporaria which regenerated following limb-bud
removal (Series I). Vicotria blue staining./ = femur; / / = tibia and fibula; c = calcaneum; a = astragalus; 12 3 4 5 = digits.
Fig. 3. A perfect hind limb of Xenopus laevis which regenerated following limb-bud
removal (Series I) .Victoria blue staining. See Fig. 2 legend for symbols.
Fig. 4. A limb of Xenopus which regenerated after limb-bud removal. By comparing
this with Fig. 3 it can be seen that the tibia and fibula are missing.
Fig. 5. Another limb of Xenopus which regenerated after limb-bud removal. Here
there is a femur, fibula, calcaneum and digits 3, 4 and 5, i.e. a posterior half limb.
Anuran limb-bud development
CN
247
248
M. MADEN
angles to the proximodistal axis. To avoid confusion they are not included in
Table I.
Series II. Distal to proximal transpositions
For these experiments, the slightly later-stage limb buds were used (Fig. 1 b and
d), from which thick slices were removed by making two cuts (at B and C),
removing the slice and replacing the tip on the proximal stump. In this fashion at
least 50 % of the mass of the exposed limb bud was removed. Although the
precise level of the cuts was not known beforehand, it can be deduced from the
cases described below where no intercalation occurred that the proximal cut
was at the level of the mid-distal femur and the distal cut at the ankle. Thus the
tissue removed was at least all of the zeugopodium.
A total of 50 Rana limb buds were so treated, 21 of which (42%, Table I)
produced perfect 5-digit limbs (see Fig. 2) despite the removal of such a large
amount of tissue. Since the limb buds were frequently examined to ensure that
the grafted tip remained in place we can conclude that these limbs exhibited
perfect intercalary regulation. The remaining 29 limbs showed various degrees
of defect which were grouped into two categories (Table 1). Thirteen (26 %) had
gross proximodistal deletions with the complete zeugopodium missing. The
other 16 (32%) were classified as 'attempted intercalation' because they had all
the segments present but with some (either the calcaneum and astragalus or
tibia and fibula) shorter than normal (Fig. 6).
Again, this ability of Rana to regulate for tissue removal is not shown by
Xenopus, where every one of the ten limbs operated upon revealed proximodistal deletions with whole segments missing (Table I). In all cases the calcaneum,
astragalus and 5 digits were present and these abutted onto the humerus;
occasionally the distal ends of the tibia and fibula were present too (Fig. 7). In
no case was there any sign of attempted intercalation of the zeugopodium.
Series III. Proximal to distal transpositions
Whole limb buds at the later stage (Fig. 1 b and d) severed at level C were
grafted to distal levels (level B). In Rana a total of 20 such operations were
performed. In all but one case the grafted limb bud did not develop well and
Fig. 6. One result of grafting a distal tip of a Rana limb bud onto a more proximal
level (Series II). Here no whole segments are missing as in many other cases (e.g.
Fig. 7 for Xenopus), but there is a defect at the ankle. This was classified as attempted
intercalation.
Fig. 7. The result of distal to proximal grafting on Xenopus (Series II). No limbs intercalated and here the very distal end of the tibia and fibula are present (//) and these
join directly onto the mid femur (/) level.
Fig. 8. The result of grafting a whole limb bud onto distal levels (Series III) in Xenopus.
Here there are two limbs tandemly repeated in the sequence femur, tibia and fibula,
femur, tibia and fibula, calcaneum and astragalus and digits.
Anuran limb-bud development
I
249
250
M. MADEN
remained as a miniature whole or part limb on the end of the host limb (at the
level of the calcaneum and astragalus). Only that one limb developed well-grown
repeated segments. The reason for the lack of development of the graft is not
clear since there were no obvious problems in matching the two cut surfaces
during the operations. This is the only series out of the several hundred operations
reported here in which this phenomenon was noted. It is thus less likely that this
can simply be attributed to incomplete healing and incorporation of the graft,
perhaps a more profound developmental reason is the cause.
With Xenopus, on the other hand, although only five buds were operated upon
each produced good limbs with fully grown repeated segments (Fig. 8). The
sequence of elements which resulted was femur, tibia and fibula (host), femur,
tibia and fibula and foot (graft).
Series IV. Controls
For this and all the subsequent series the earlier stage limb buds were used
(Fig. 1 a and c).
In this series the limb buds were amputated at level A and replaced in the same
orientation to serve as controls for the following axial inversion series. In Rana
20 limb buds were so treated, 17 of which produced perfect 5-digit limbs (see
Fig. 2). The remaining three displayed curved and shortened tibiae and fibulae, a
phenomenon seen frequently throughout the following series. One of these three
also has one extra digit.
In Xenopus ten control limb buds were operated upon and all produced perfect 5-digit limbs (see Fig. 3) without any signs of the level of the cut being
apparent.
Series V. Dorsoventral inversion
By grafting left limb buds onto right stumps, the dorsoventral axis can be
inverted whilst the anteroposterior axis remains normally oriented. This
operation resulted in a high proportion of supernumerary limb induction in
Rana (Table 2). Of the 28 operated limbs 17 produced supernumeraries (61 %),
15 of which were double (Fig. 9) and 2 single. They bifurcated mostly at the knee,
but occasionally higher in the femur or lower at the ankle. Of the total of 32
supernumeraries 78 % had 4 or 5 digits and all of these were of stump handedness
(right). The position of origin of these supernumeraries was rather confusing.
Whilst the majority arose at the dorsal or ventral poles, some were displaced into
adjacent quadrants. Surprisingly, four limbs had supernumeraries which arose at
the anterior or posterior poles of the host limb; perhaps this was caused by the
rotation of the graft after the operation.
In Xenopus, by contrast, the frequency of supernumerary induction was much
lower, being only 15 % (3 out of 20). Each was a single supernumerary, which
bifurcated in the femur, two at the dorsal pole and one at the ventral pole. Two
Fig. 9. Dorsoventral inversion (Series V) of Rana limb buds. From the ventral pole of the limb arises a 5-digit right-handed
supernumerary (Sj), but with some phalanges missing. In the middle is the left limb bud (G) which was grafted onto the right
stump. From the dorsal pole of the limb arises a perfect 5-digit right-handed supernumerary (S2). Both supernumeraries are
thus of stump handedness and bifurcate at the knee.
Fig. 10. Dorsoventral inversion of Xenopus limb buds (Series V). Here only single supernumeraries (S) were produced. This
one was a perfect 5-digit limb of stump handedness arising at the ventral pole.
10
X :.*-«••
to
•T
8-
I
252
M. MADEN
•.
11
12
*
253
Anuran limb-bud development
Table 2. Summary of the results of the three types of axial inversions on the limb
buds of axolotl {data from Maden & Goodwin, 1980), Rana, Xenopus {results
reported here) and chick {data from Saunders, Gasseling & Gfeller, 1958) assembled
into one table for comparative purposes
Animal
Axolotl
Operation
Number of
limbs with Number of Supernumeraries
Super- r
numeraries Single
Double
Triple
(%)
(%)
(%)
(%)
Chick
DV
DV
DV
DV
inversion
inversion
inversion
inversion
40
61
15
19?
60
12
100
100
40
88
0
0
0
0
0
0
Axolotl
Rana
Xenopus
Chick
AP
AP
AP
AP
inversion
inversion
inversion
inversion
70
54
30
65
65
21
100
91
35
79
0
9
0
0
0
0
Axolotl
Rana
Xenopus
Chick
APDV inversion
APDV inversion
APDV inversion
APDV inversion
38
67
20
70
83
46
75
84
17
41
25
16
0
13
0
0
Rana
Xenopus
were perfect 5-digit limbs of stump handedness (Fig. 10) and one had only the
posterior 3 digits. This contradicts the report by Cameron & Fallon (1977) that
Xenopus does not produce DV supernumeraries.
Series VI. Anteroposterior inversion
Grafting left limb buds onto right stumps in normal dorsoventral orientation
results in the inversion of the anteroposterior axis. In Rana, this operation resulted in a good frequency of induction of supernumeraries - 14 out of 26 limbs or
54 % (Table 2). As in the previous series the vast majority of these (11 in all) were
double supernumeraries (Fig. 11) and the remaining three, single. Again they
bifurcated in the vicinity of the knee. The majority (22 out of 25) were well
Fig. 11. An anteroposterior inversion (Series VI) of Rana limb buds which resulted
in two supernumeraries. At the anterior position is a right-handed supernumerary
(Sx), then the grafted left limb, then at the posterior position a right handed supernumerary (S2). The digital sequence from top to bottom is 1 2 3 4 5 (Sj) 5 4 3 21
(graft) 12 3 4 5 (S2). Thus both supernumeraries are of stump handedness.
Fig. 12. Anteroposterior inversion of Xenopus limb buds (Series VI). Here a 4-digit
right-handed supernumerary (S) appeared at the anterior pole of the grafted left limb
bud (G).
,j
i M H ft 3
254
M. MADEN
formed supernumeraries with either 4 or 5 digits and all of these were of stump
handedness. Most of them arose around the anterior or posterior poles of the
host limb, but four limbs produced supernumeraries at the dorsal or ventral
poles which, as in the previous series, confused the issue somewhat. Further
complicating the situation was one peculiar supernumerary which was double
anterior in structure.
In Xenopus a lower frequency was produced - 30 % (6 out of 20 - Table 2). All
were single as in the DV Xenopus series and all except one appeared at the
anterior pole. Only two were good 4-or 5-digit limbs (Fig. 12).
Series VII. Dorsoventral arid anteroposterior inversion
In this series limb buds were amputated and replaced upside down, thus
reversing both limb axes. In Rana, a total of 58 such operations were performed
and 39 of these (67 % - Table 2) produced supernumerary limbs which were
remarkable for two phenomena. Firstly, eleven supernumeraries were double
posterior limbs. They did not arise in any consistent location with respect to the
host axes, they could be either single or present in conjunction with another
normal supernumerary (two double posteriors were never produced) and had
either 3, 4 or 5 digits (Figs. 13 and 14). Secondly, five cases of triple supernumeraries arose. Fig. 15, for instance, is a right limb which has a total of 18
digits. It consists of the grafted right limb, a ventral left-handed supernumerary, a posterodorsal right-handed supernumerary and a posterodorsal
left-handed supernumerary.
The remaining limbs produced approximately equal numbers of either single
or double supernumeraries (Fig. 16) from all quadrants of the host limb - no
particular position of origin was preferred. As before, most bifurcated at the
Fig. 13. A double posterior supernumerary (S) resulting from APDV inversion of
Rana limb buds (Series VII). It arose at the posterior pole and has a mirror-imaged
calcaneum (C) and a digital sequence of 5 4 4 5. Above, is the rotated left limb bud.
Fig. 14. A double posterior supernumerary (S) resulting from APDV inversion of
Rana limb buds (Series VII). It has two calcanea, a digital sequence of 5 4 3 4 5, and
arose in the posterior-ventral quadrant. Above is the rotated left limb.
Fig. 15. A triple supernumerary resulting from APDV inversion of Rana limb buds
(Series VII). The limb has a total of 18 digits. In the middle is the rotated right limb
(G). To the left is a 5-digit left-handed supernumerary (Sx) which arose at the ventral
pole. To the right is a V-shaped double supernumerary (S2), one left-handed and one
right-handed with their ventral surfaces facing. There are two astragali (a), a central
fused calcaneum and 8 digits with digits 4 and 5 being fused. This arose in the posterodorsal quadrant.
Fig. 16. A classical double supernumerary from APDV inversion of Rana limb buds
(Series VII;. In the middle is the right graft (G). Above is a 5-digit left-handed supernumerary (SO in the posteroventral quadrant and below is a 5-digit right-handed
supernumerary (S2) in the anteroventral quadrant, thus making a pair of opposite
handedness.
Anuran limb-bud development
255
CD
256
M. MADEN
17
\
18
Anuran limb-bud development
257
knee. Where they could be identified, pairs of supernumeraries were always
found to be of opposite handedness. Many, however, were unidentifiable since
the digits did not curve. It is possible that these could be mirror-imaged in the
dorsoventral plane as has been found in regenerating axolotl limbs after 180°
rotation (Maden, 1980/3). This is currently being investigated by studying the
muscle patterns in serial sections.
In Xenopus, rotating the limb bud 180° resulted in a low frequency of induction
of 20 % (4 out of 20, Table 2). One of these was a double supernumerary (Fig.
17), the other three single (Fig. 18). They bifurcated at the knee or higher, only
one was a perfect 5-digit supernumerary (of opposite handedness to the stump),
one was a 4-digit limb and the other three only developing 2 digits. No structures
resembling the double posterior supernumeraries of Rana were produced.
Many of the rotated limbs displayed proximodistal deletions at the level of
rotation with, for instance, the distal part of the femur or the proximal tibia and
fibula missing. In Fig. 18 the calcaneum and astragalus of the 4-digit supernumerary abut directly onto the knee of the rotated limb. This is in contrast to
the other two rotation series (V and VI) where the level of rotation could only be
detected by a sudden inversion of limb structure, rather than by deleted tissue.
Thus, this type of defect seems to be related to the complete inversion of limb
buds.
DISCUSSION
The proximodistal axis
When the limb buds of axolotls (Maden & Goodwin, 1980) and other
Urodele species (Harrison, 1918) are removed a perfect limb nevertheless
develops. On the other hand there is universal agreement (Lillie, 1904; Shorey,
1909; Peebles, 1911; Spurling, 1923; Saunders, 1948; Hampe, 1959) that the
chick limb produces truncations following such an operation. Between these two
extremes are the Anurans. Rana temporaria almost always produced perfect
limbs here and earlier stages of other species of Rana do likewise (Byrnes, 1898).
But Xenopus laevis produced perfect limbs in less than half the cases (Table 1).
This same gradation in regulative ability is evident in the results of distal to
proximal grafts (Series II) in which a large slice of the limb bud is removed.
Fig. 17. A double supernumerary resulting from APDV rotation of Xenopus limb
buds. The grafted left limb bud (G) is still upside down. A perfect 5-digit right-handed
supernumerary (Sx) arose in the posterodorsal quadrant and a posterior half 2-digit
supernumerary (S2) arose in the anterodorsal quadrant.
Fig. 18. A single 4-digit supernumerary resulting from APDV rotation of Xenopus
limb buds (Series VII). Note the calcaneum and astragalus of the supernumerary (S)
abut directly onto the proximal end of the tibia and fibula of the rotated limb producing a considerable proximodistal disparity in levels.
258
M. MADEN
Axolotls undergo perfect intercalary regulation (Maden & Goodwin, 1980), the
chick limb bud produces limbs with severe deletions (Summerbell, 1977; Kieny
& Pautou, 1977) and in between are the Anurans. Rana undergoes intercalation
in just under half the cases, but Xenopus does not at all (Table 1). The converse
experiment, however, that of grafting a whole limb bud to distal levels (Series
III) produces uniformity of results - serially repeated limbs are the norm (Table
1).
Not only do these different species vary in their responses to the experiments
described above, but in Xenopus and Rana, which are of intermediate regulative
ability, even members of the same species do not behave consistently. For
instance, in Rana, after distal to proximal grafts some limbs intercalated perfectly, some did so partially and yet others did not at all. This phenomenon
further confounds the search for general rules of development.
An important conclusion which emerges from this study of Anuran limb
development is that the ability to regulate for limb-bud amputation is not related to the ability to regulate for intercalary deletions. If the two were strictly
dependent then Rana, which always regenerates the limb bud, should always
replace such intercalary deletions, but the latter only occurs in less than half
the cases. Conversely, Xenopus which regenerates limb buds in 38 % of the cases
should be able to intercalate deletions in a certain proportion, but it cannot. In
further support of this conclusion the Xenopus distal to proximal grafts of Series
II were performed on right limb buds while the left limb buds of the same
animals were simply amputated at the proximal level. In several animals the left
limb buds regenerated complete limbs yet their contralateral partners did not
intercalate for proximodistal deletions.
As an explanation of this unrelatedness it is possible that the regeneration of
limb buds is primarily a property of the epidermis whereas regulation for
proximodistal deletion is a property of the mesoderm. Regeneration of limb buds
would thus depend on whether or not epidermis can close the wound, and reinitiate
developement. Indeed, the terminal defects found after limb-bud amputation
can be duplicated in each of these organisms by permanently removing the
epidermis {Triton - Balinsky, 1935; Xewo/m-Tschumi, 1957; chick - Saunders
1948; Summerbell, 1974.) Although the epidermis rapidly heals over the cut limb
bud in chick, the apical ectodermal ridge (AER) is not regenerated (Saunders,
1948). To explain the results reported here on this basis we must assume that the
AER is only regenerated in a certain percentage of cases in Xenopus and always
in Rana, a suggestion which can easily be tested by performing the relevant
histology.
The latter case, that regulation for proximodistal deletion is a property of the
mesoderm, seems highly likely since this phenomenon takes place between two
cut edges of mesoderm a long way from the ectodermal covering at the tip of the
limb bud. Therefore we can conclude that axolotl, Rana, Xenopus and chick
display decreasing regulative ability (or increasing mosaicism) in the meso-
Anuran limb-bud development
259
derm of the limb bud (Table 1), at least as far as the proximodistal axis is
concerned.
What is the significance of these divergent properties of vertebrate limbs for
current models of development in the proximodistal axis? One model, developed
from studies using the chick limb, is the progress zone model (Summerbell,
Lewis & Wolpert, 1973). It hypothesises that change in positional value takes
place within the progress zone at the tip of the limb bud and outside, the cells
rapidly lose this ability. It can very satisfactorily explain the mosaic results, that
is chick limb-bud removal, proximodistal deletion experiments in the chick and
Xenopus, and the proximodistal deletion observed after limb-bud amputation in
Xenopus (Table 1). But this model cannot in its present form be modified to
describe the regulative behaviour of axolotl and Rana limb buds. On the other
hand an averaging model such as that developed for the regenerating adult limb
(Maden, 1977) is highly regulative and can explain very well the normal proximodistal sequence of limb development (Maden, unpublished), the varied results of
limb-bud amputation (provided this depends on the regeneration or not of the
apical epidermis) and the proximodistal intercalation of axolotl and Rana. But
the problem with this model lies in preventing interaction between neighbouring
cells and so mosaic behaviour cannot be explained unless other assumptions are
made. One solution is to use a model with two cell-state parameters rather than
just one as in the model above, and vary one of them from species to species, or
in the case of the Anurans, particularly Rana, vary them from individual to
individual (Meinhardt & Gierer, 1980; Summerbell, unpublished).
The transverse axes
In contrast to the increasing mosaicism of axolotl, Rana, Xenopus and chick
limb buds in the proximodistal axis, experiments on the transverse axes reveal a
dramatic uniformity of results across these vertebrate groups. With one possible
exception (dorsoventral inversion of the chick limb) the net effect of inversion of
one or both transverse axes is to produce supernumerary limbs. The variables
concern the number of supernumeraries, their frequency, position and structure
(Table 2).
DV inversion. Frequency: After dorsoventral inversion Rana produces the
highest frequencies of supernumeraries, nearly all being double, followed by
axolotl and Xenopus. The low frequency recorded in Table 2 for chick refers to the
occurrence of bidorsal and biventral wings after such an operation (Saunders,
Gasseling & Gfeller, 1958). Similar structures are occasionally observed in
regenerating limbs and could represent a supernumerary fused with the inverted graft. However, general opinion seems to be that chick limbs do not produce supernumeraries after dorsoventral inversion. Whether this is due to
absence of outgrowth because there is no AER on the dorsal or ventral surfaces
or because of a lack of interaction in the mesoderm is unknown.
260
M. MADEN
Position: Supernumeraries appear either at dorsal or ventral poles with singles
showing no distinct preference for either position. In axolotl and Rana some are
found at anterior and posterior positions but in these cases the orientation of
the graft had changed. Structure: In all cases where handedness can be determined
DV supernumeraries are of stump handedness.
These observations force us to consider how the dorsoventral axis is organized
during development, a subject which has received very little attention compared
to the anteroposterior axis. The few experiments on the chick limb (Pautou &
Kieny, 1973; McCabe, Errick & Saunders, 1974) have merely concluded that
dorsoventral polarity resides in the mesoderm, but can be modified by the
ectoderm during certain phases of limb development. However, there exists
enough evidence from studies on much earlier stage Urodele embryos to suggest
that there is a dorsal organizer analogous to the more well-known posterior
organizer which behaves in the same fashion following axial reversal (Hollinshead,
1936; Swett, 1938). The presence of both regions is obligatory for full and complete development of the limb, as recently shown by Slack (1980).
The experiments on the dorsoventral axis described here are perfectly adequately explained by the hypothesis that there is a dorsal organizer which is the
source of a diffusible morphogen with properties analogous to, yet distinct from,
the posterior organizer (Slack, 1977*7,6; Tickle, Summerbell &Wolpert, 1975).
A totally different concept, yet equally competent to explain these results, is the
polar coordinate model (French, Bryant & Bryant, 1976) which considers the
transverse axes of the limb to be a single circumferential field rather than two
orthogonal gradients. We shall see below how these two hypotheses fare in
explaining the remaining results.
AP inversion. Frequency: Here, the axolotl has the highest frequency of supernumeraries closely followed by the chick and Rana, with Xenopus again the least
frequent. All except Xenopus can produce double supernumeraries, with Rana
again invariably doing so. Position: The position of single supernumeraries
varies along with their frequency. In Xenopus most singles are at the anterior pole,
in axolotl either anterior or posterior and in chick and Rana at the posterior pole.
Double supernumeraries are, of course, at both anterior and posterior poles, but
some in Rana and axolotl are found at dorsal and ventral positions. As above, in
these cases the orientation of the graft had changed. Structure: In all cases where
handedness can be determined AP supernumeraries are of stump handedness.
This, as in the previous series seems to be a general rule.
The organization of the anteroposterior axis has been the subject of extensive
investigation and theorizing, with the greatest emphasis being on the concept of
a posterior zone as a source of a diffusible morphogen which is responsible for
organization in this axis (Tickle et al. 1975; Slack, 1977a, b). More recently, the
polar coordinate model (French et al. 1976) has become an alternative description, and has, in particular, been applied to the wealth of data on the chick limb
bud (Iten & Murphy, 1980). As in the previous series,both hypotheses areequally
capable of explaining these results on the inversion of the anteroposterior axis.
Anuran limb-bud development
261
APDV inversion. Frequency: After rotating limb buds 180° to reverse both
axes, the chick and Rana have the highest frequencies of induction of Supernumeraries, followed by the axolotl and, again, Xenopus being least frequent.
However, Cameron & Fallon (1977) reported a much higher figure for Xenopus 76 % - which would put it ahead of all four organisms. Each can produce
single or double supernumeraries but most significant are several cases of triple
supernumeraries in Rana. These are also occasionally seen in regenerating
axolotl limbs (Maden, unpublished) and in the stick insect (Bart, 1971). In the
latter, triple supernumeraries formed in almost an identical number of cases
(14 vs 13% here). Position: The axolotl shows a distinct preference for supernumeraries to appear in the anteroventral quadrant, although they do appear in
other positions as well. In the chick the majority formed at the anterior and
posterior poles, but 10% arose at the dorsal or ventral poles. In Rana and
Xenopus neither seem to show a preference for any particular position although
Cameron & Fallon (1977) noted that supernumeraries only arose in Xenopus
at the anterior or posterior poles. It is difficult to draw any general conclusions
from this data. Structure: In the chick two of the seven pairs of supernumeraries
generated were of the same handedness as each other and the remaining five
pairs were opposite. It is very interesting to note that although their bone
structure appeared normal, the feather patterns were mirror imaged, a phenomenon that only occurred after 180° rotation and not after AP inversion (Saunders,
et al., 1958). These supernumeraries may not be normal in the dorsoventral axis.
In the axolotl the supernumeraries were not of good enough quality to determine
handedness. Some had digits that did not curve and so could have been mirror
imaged in the dorsoventral axis as has been found in regenerating limbs of
axolotls (Maden, 1980a). So, too, in Xenopus where most supernumeraries
looked normal after cartilage staining, but in a few the digits did not curve.
Rana broke all the rules. Of the five triple supernumeraries, the handedness of
four of them could not be completely determined because of straight digits.The
one that could had two limbs of the same handedness as the stump and the other
opposite. Two of the double supernumeraries whose structure could be determined were of the same handedness as each other and six of them were opposite
Eleven supernumeraries were double posterior of varying degrees of mirror
imaging and could appear either on their own or accompanied by a normal
supernumerary. And in addition many were of indeterminate handedness
because their digits did not bend and thus could have been mirror imaged in the
dorsoventral plane.
The only general conclusion to emerge from this analysis is that APDV
supernumeraries can arise in many positions, can be of normal or mirror imaged
structure and perhaps the plane of mirror imaging is species dependent. However, what is quite clear is that the situation after APDV inversion is much more
complicated than after AP or DV inversion.
262
M. MADEN
Diffusion models which, in their present state of development concentrate
only on the anteroposterior axis, are quite incapable of explaining the above
results. This is because an APDV inversion is not equivalent to an AP inversion
since the resultant structures are vastly different. Thus a double-gradient model
needs to be developed and its detailed behaviour tested to determine whether it
can match this diversity of experimental results.
On the other hand, the polar coordinate model goes a considerable way to
explaining these results. The occurrence of triple supernumeraries and the lack of
constant positions of origin suggests that the original evenly spaced form of the
clockface adopted for the insect leg is more appropriate. Pairs of supernumeraries should, however, be of opposite handedness, which they are not necessarily. Mirror-imaged structures are explicable by this model, but specific
additional predictions appear as unfortunate side effects. These are that supernumeraries mirror imaged in the anteroposterior axis should appear only at the
dorsal and ventral poles, mirror images in the dorsoventral axis should appear
only at the anterior and posterior poles and that mirror images should also
appear at a certain frequency after AP and DV inversions. These predictions do
not appear to hold, but perhaps additional assumptions can be built into the
model to facilitate a better fit to the experimental findings.
Development and regeneration
What is immediately apparent from the above analysis is how remarkably
similar the results of most of these experiments on developing limb buds are to
those on regenerating limbs. In the transverse axes, for instance, DV inversion
gives supernumerary limbs of the same handedness as the stump at dorsal or
ventral poles (Tank, 1978; Wallace & Watson, 1979; Maden, 1980a) as does AP
inversion, but at anterior or posterior poles (Iten & Bryant, 1975; Tank, 1978;
Wallace & Watson, 1979; Maden, 1980a). 180° rotation gives supernumeraries
of variable structure, either normal, double-dorsal or double-ventral in various
combinations (Maden, 1980a). Although the precise structure of supernumeraries from 180° limb-bud rotations has not yet been examined it is possible that
they too are abnormal in this respect as noted above. Thus we can conclude that
as far as the transverse axes are concerned development and regeneration seem
to be governed by the same developmental rules. It is thus valid to attempt to
produce theories which not only describe vertebrate limb development, but
regeneration as well (e.g. French et al. 1976).
However, this uniformity is not the case in the proximodistal axis. Only
axolotl limb buds behave in the same fashion as regenerating limbs which undergo perfect intercalary regulation after distal to proximal shifts (Iten & Bryant,
1975; Stocum, 1975; Maden, 19806). Rana, Xenopus and chicks are increasingly
less regulative in this axis, which confounds the search for generalised rules of
vertebrate limb development.
Anuran limb-bud development
263
With these conclusions in mind it is interesting to consider whether organisms
that regenerate their limbs in adult life show any consistent differences in developmental behaviour from those that cannot regenerate. In the transverse axes this
is not the case since all seem to behave in a similar fashion during development.
But in the proximodistal axis there is a correlation. Axolotls regenerate throughout their life and show complete regulation in the proximodistal axis. Rana
temporaria can regenerate for a short period after limbs have developed
(Polezhayev, 1946) and show intermediate regulation. Xenopus loses regenerative
ability before limb development has terminated (Dent, 1962) and is mosaic in the
proximodistal axis. Finally, chick limbs, which can never regenerate, are the
most mosaic of all. Therefore, the capacity of adult animals to regenerate limbs
reflects the persistence of embryonic regulative properties throughout the
developmental period and into the adult form, with no transition to the mosaic
condition occurring. Stimulation of limb regeneration in mammals would thus
require a reawakening of the regulative capacity, which may be impossible if
mosaicism results from a state of the mesoderm, rather than a deficiency in the
epidermis or the lack of some systemic factor.
I would like to thank Katriye Mustafa for excellent technical work throughout this study
and Dennis Summerbell for putting me right during many discussions on the manuscript.
REFERENCES
B.I. (1935). Selbstdifferenzierung des Extremitatenmesoderms im Interplant.
Zool. Jb. (Allgemeine Zoologie u.Physiologie der Tiere) 54, 327-348.
BART, A. (1971). Morphogenese surnumeraire au niveau de la patte du phasme Carausius
morosus Br. Wilhelm Roux Arch. EntwMech. Org. 166, 331-364.
BYRNES, E. F. (1898). On the regeneration of limbs in frogs after the extirpation of limbrudiments. Anat. Anz. 15, 104-107.
BYRNES, E. F. (1904). Regeneration of the anterior limbs in the tadpoles of frogs. Wilhelm
Roux Arch. EntwMech. Org. 18, 171-177.
CAMERON, J. & FALLON, J. F. (1977). Evidence for polarizing zone in the limb buds of Xenopus laevis. Devi Biol. 55, 320-330.
DENT, J. N. (1962). Limb regeneration in larvae and metamorphosing individuals of the
South African clawed toad. /. Morph. 110, 61-77.
EDE, D. A., HINCHLIFFE, J. R. & BALLS, M. (1977). Vertebrate limb and somite morphogenesis.
Symp. Br. Soc. Devi Biol. no. 3 Cambridge University Press.
FRENCH, V., BRYANT, P. J. & BRYANT, S. V. (1976). Pattern regulation in epimorphic fields.
Science 193, 969-981.
HAMPE, A. (1959). Contribution a l'etude du developpement et de la regulation des deficiences
et des excedents dans la patte de l'embryon de poulet. Archs Anat. microsc. Morph. exp. 48,
345-478.
HARRISON, R. G. (1918). Experiments on the development of the forelimb of Amblystoma,
a self-differentiating equipotential system. /. exp. Zool. 25, 413-461.
HARRISON, R. G. (1921). On relations of symmetry in transplanted limbs. /. exp. Zool. 32,
1-136.
HOLLINSHEAD, W. H. (1936). Determination of the dorsoventral axis of the fore limb in
Amblystoma tigrinum. J. exp. Zool. 73, 183-194.
ITEN, L. E. & BRYANT, S. V. (1975). The interaction between the blastema and stump in the
establishment of the anterior-posterior and proximo-distal organisation of the limb
regenerate. Devi Biol. 44, 119-147.
BALINSKY,
264
M. MADEN
L. E. & MURPHY, D. J. (1980). Pattern regulation in the embryonic chick limb: supernumerary limb formation with anterior (non-ZPA) limb bud tissue. Devi Biol. 75, 373-385.
KIENY, M. & PAUTOU, M-P. (1977). Proximo-distal pattern regulation in deficient avian limb
buds. Wilhelm Roux' Arch, devl Biol. 183, 177-191.
LILLIE, F. R. (1904). Experimental studies on the development of organs in the embryo of the
fowl (Gallus domesticus). Biol. Bull. mar. biol. Lab., Woods Hole. 7, 33-54.
MADEN, M. (1977). The regeneration of positional information in the amphibian limb.
/. theor. Biol. 69, 735-753.
MADEN, M. (1980a). Structure of supernumerary limbs. Nature 287, 803-805.
MADEN, M. (19806). Intercalary regulation in the amphibian limb and the rule of distal
transformation. J. Embryol. exp. Morph. 56, 201-209.
MADEN, M. & GOODWIN, B. C. (1980). Experiments on developing limb buds of the axolotl
Ambystoma mexicanum. J. Embryol. exp. Morph. 57, 177-187.
MACCABE, J. A., ERRICK, J. & SAUNDERS, J. W. (1974), Ectodermal control of the dorsoventral axis in the leg bud of the chick embryo. Devi Biol. 39, 69-82.
MEINHARDT, H. & GIERER, A. (1980). Generation and regeneration of sequence of structures
during morphogenesis. /. theor. Biol. 85, 429-450.
NIEUWKOOP, P. D. & FABER, J. (1967). Normal Table o/Xenopus laevis (Daudin). Amsterdam:
North-Holland.
PAUTOU, M-P. & KIENY, M. (1973). Interaction ecto-mesodermique dans l'etablissement de
la polarite dorso-centrale du pied de l'embryon de poulet. C. r. hebd. Seanc. Acad. Sci.,
Paris D 277, 1225-1228.
PEEBLES, F. (1911). On the interchange of limbs of the chick by transplantation. Biol Bull
mar. biol. Lab., Woods Hole 20, 14-18.
POLEZHAYEV, L. W. (1946). The loss and restoration of regenerative capacity in the limbs of
tailless Amphibia. Biol. Rev. pp. 141-147.
SAUNDERS, J. W. (1948). The proximo-distal sequence of origin of the parts of the chick wing
and the role of the ectoderm. /. exp. Zool. 108, 363-403.
SAUNDERS, J. W., GASSELING, M. T. & GFELLER, M. D. (1958). Interactions of ectoderm and
mesoderm in the origin of axial relationships in the wing of the fowl. /. exp. Zool. 137,
39-74.
SHOREY, M. L. (1909). The effect of the destruction of peripheral areas on the differentiation
of the neuroblasts. /. exp. Zool 7, 25-64.
SLACK, J. M. W. (1977a). Determination of anteroposterior polarity in the axolotl forelimb
by an interaction between limb and flank rudiments. /. Embryol. exp. Morph. 39, 151-168.
SLACK, J. M. W. (19776). Control of anteroposterior pattern in the axolotl forelimb by a
smoothly graded signal. /. Embryol. exp. Morph. 39, 169-182.
SLACK, J. M. W. (1980). Regulation and potency in the forelimb rudiment of the axolotl
embryo. J. Embryol exp. Morph. 57, 203-217.
SPURLING, R. G. (1923). The effect of extirpation of the posterior limb bud on the development
of the limb and pelvic girdle in chick embryo. Anat. Rec. 26, 41-56.
STOCUM, D. L. (1975). Regulation after proximal or distal transposition of limb regeneration
blastemas and determination of the proximal boundary of the regenerate. Devi Biol 45,
112-136.
SUMMERBELL, D. (1974). A quantitative analysis of the effect of excision of the AER from the
chick limb-bud. /. Embryol exp. Morph. 32, 651-660.
SUMMERBELL, D. (1977). Regulation of deficiences along the proximal distal axis of the chick
wing bud: a quantitative analysis. J. Embryol. exp. Morph. 41, 137-159.
SUMMERBELL, D. & LEWIS, J. H. (1975). Time, place and positional value in the chick limb-bud
/. Embryol. exp. Morph. 33, 621-643.
SUMMERBELL, D., LEWIS, J. H. & WOLPERT, L. (1973). Positional information in chick limb
morphogenesis. Nature 244, 492-496.
SWETT, F. H. (1927). Differentiation of the amphibian limb. /. exp. Zool 47, 385-439.
SWETT, F. H. (1938). Experiments upon the relationship of surrounding areas to polarization
of the dorsoventral limb-axis in Ambystoma punctatum (Linn.). /. exp. Zool 78, 81-100.
ITEN,
Anuran limb-bud development
265
TANK, P. W. (1978). The occurrence of supernumerary limbs following blastemal transplantation in the regenerating forelimb of the axolotl, Ambystoma mexicanum. DevlBiol, 62,
143-161.
TAYLOR, A. C. & KOLLROS, J. J. (1946). Stages in the normal development of Rana pipiens
larvae. Anat. Rec. 94, 7-23.
TICKLE, C , SUMMERBELL, D. &WOLPERT, L. (1975). Positional signalling and specification of
digits in chick limb morphogenesis. Nature 254, 199-202.
TSCHUMI, P. A. (1957). The growth of the hindlimb bud of Xenopus laevis and its dependence
upon the epidermis. J. Anat. 91, 149-173.
WALLACE, H. & WATSON, A. (1979). Duplicated axolotl regenerates. J. Embryol. exp. Morph.
49, 243-258.
WARREN, A. E. (1934). Experimental studies on the development of the wing in the embryo of
Gallus domesticus. Am. J. Anat. 54, 449-485.
{Received 9 October 1980, revised 10 December 1980)