J. Embryol. exp. Morph. 76, 177-197 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Size dependence during the development of the
amphibian foot. Colchicine-induced digital loss and
reduction
By P. ALBERCH AND EMILY A. GALE
From the Museum of Comparative Zoology, Harvard University,
Massachusetts
SUMMARY
Localized treatment of the limb buds of the frog, Xenopus laevis, and the salamander,
Ambystoma mexicanum, with the mitotic inhibitor colchicine results in limbs that, when
compared with the contralateral control, are smaller in size and have lost skeletal elements.
There is a very well defined pattern in terms of what elements are most likely to be lost. For
example, frogs that have lost a toe always lose the first toe, while salamanders always lose the
fifth. These differences correspond to qualitative differences in developmental sequence of
digital differentiation in anurans as compared to urodeles. We propose a hypothesis in which
the digital pattern is indirectly affected by reduction in the number of mesenchymal cells in
the embryonic field.
INTRODUCTION
In 1949 Bretscher reported a series of experiments which showed that local
treatment of the early limb bud of the frog Xenopus laevis with the mitotic
inhibitor colchicine resulted in the reduction, and even loss, of digits. These
experiments were further elaborated and discussed in Bretscher & Tschumi
(1951) and Tschumi (1953; in this paper the development was inhibited using
chloroethylamine). These authors interpreted their results in terms of competition for cells among the various skeletal elements during chondrogenesis.
These competitive interactions, again according to Bretscher & Tschumi, in a
limb bud with an artificially reduced number of cells resulted in some welldefined patterns of digital reduction. These regularities were particularly evident
in the fact that some digits were affected more frequently than others. For
example, digits 3 and 4 were very rarely affected, while digit 1 (the 'thumb') was
always the most reduced. Based on that information, they pointed out the potential phylogenetic implications of their results and drew an analogy between their
experimentally observed patterns of digital reduction and loss and the phyletic
trend toward limb reduction observed in some groups of reptiles and mammals.
1
Author's address: Museum of Comparative Zoology, Harvard University, Cambridge,
Massachusetts 02138, U.S.A.
178
P. ALBERCH AND E. A. GALE
(At that time, Bretscher & Tschumi did not know of any anuran which had lost
either a complete digit or single phalanges.) Rensch (1959), Waddington (1962)
and Devillers (1965) reported Tschumi's results and their potential implications
for evolutionary studies. However, their work has been mostly forgotten and it
has had very little impact in the literature on evolution or development.
Nevertheless, it was an appealing system in which any developmental perturbation that resulted in a limb bud with a smaller number of primordial cells could
indirectly result in the loss or reduction of digital elements. This evolutionary
issue is dealt with in a related paper (Alberch & Gale, 1983).
These experiments also posed some interesting developmental questions
deserving closer scrutiny. One, in particular, is the relationship between size and
pattern formation in embryonic processes. Most theoretical models of pattern
formation (e.g. Turing, 1952; Kauffman, Shymko & Trabert, 1978; Newman &
Frisch, 1979; Murray, 1981; Meinhardt, 1982) show some degree of size dependence, i.e., the final pattern is dependent on the size of the embryonic field at
the time of the process. In fact, some models, like Kauffman et al. (1978) and
Newman & Frisch (1979), use tissue growth as an organizing agent in their
developmental systems. Conversely, Cooke (1979, 1981, 1982), Tarn (1981),
Maden (1981a) and Dan-Sohkawa & Sato (1978) among others have presented
experimental evidence that some pattern formation processes are scale independent. Obviously, these two views need not be mutually exclusive. Some embryonic processes might be largely scale independent while others might not.
Even the same system could be scale independent at some stage in its development and not at some other.
Bretscher & Tschumi envisioned amphibian limb development as dependent
on the size (roughly equivalent to number of undifferentiated cells) of the embryonic limb field. Other authors have also shown, since then, that the treatment
of developing limbs with various mitotic inhibitors triggers loss and/or reduction
of elements (e.g. Kieny, 1975; Scott, Ritter & Wilson, 1977; Raynaud, 1981).
Patterns similar to the ones reported by Bretscher & Tschumi resulted regardless
of the experimental taxa used (amphibians, reptiles, birds or mammals). That is,
the loss of elements was not chaotic but it exhibited well-defined regularities.
Most of these authors, however, were interested in mechanistic problems and did
not emphasize the comparative aspects of their results. Obviously not all
teratogens induce digital or phalangeal loss (see Tickle & Wolpert, 1981, for a
review on teratogenic effects in limb development). For example, Scott et al.
(1977,1980), Klein, Scott & Wilson (1978) and Scott (1981) have discussed other
drugs that induce polydactyly by affecting the timing of onset in the physiological
necrosis that characterizes the normal development in higher vertebrates. This
would not apply to our system since cell death does not appear to play a role in
amphibian limb development (Cameron & Fallon, 1911 a).
In this paper we report on a series of experiments where we modify and
expand on Bretscher & Tschumi's experimental protocol. We locally treated
Amphibian limb development
179
with colchicine the limb buds of the anuran, Xenopus laevis, and the urodele,
Ambystoma mexicanum, at various stages of limb development. Unlike
Bretscher & Tschumi, we focused on the differentiation of individual skeletal
elements rather than on external size measurements. Also, we controlled for
specific developmental stage at the time of treatment. Bretscher & Tschumi did
not specifically state when they treated their Xenopus, although their illustrations seem to indicate that they used stage-52 animals (Nieuwkoop & Faber,
1956; Fig. 1). We give a detailed quantitative account of the experimentally
induced morphologies. The experimental results corroborate the ones obtained
by Bretscher & Tschumi and pose interesting questions relating to current
models of limb morphogenesis and to qualitative differences between urodele
and anuran limb development.
MATERIALS AND METHODS
For this study, laboratory strains of Xenopus laevis (bred in our laboratory or
pre-limb-bud-stage tadpoles purchased from Carolina Biological Supply) and
Ambystoma mexicanum (eggs obtained from the Axolotl Colony, Indiana
University) were used. The axolotls were raised individually in glass jars, in 10 %
Steinberg's solution, while the Xenopus were raised in small groups in Modified
Holtfreter's Solution (50 % Holtfreter's with 0-1 g/1 MgSO 4 , 7H2O added). The
axolotls were kept separated to prevent cannibalistic leg damage thereby avoiding the digital anomalies due to regeneration.
In this experiment, both the axolotls and the Xenopus laevis were treated using
a method slightly modified after Bretscher (1949). We staged the Xenopus according to the normal table of Nieuwkoop & Faber (1956) and the axolotls based
on the number of externally visible toes. Larvae were then anaesthetized in a
1: 7000 concentration of ethyl m-aminobenzoate and placed, left side up, in a
small cavity carved into a paraffin-filled Petri dish. The head and tail of the
animal to be treated were covered with wet paper towels to prevent desiccation
but not so tightly as to cause suffocation. Under a dissecting microscope, the left
hindlimb bud was gently perforated in four evenly distributed points with a
sharpened tungsten needle. (This was necessary to help the rapid diffusion of the
colchicine into the limb bud). A small piece of tinfoil was placed under the limb
bud isolating it from the underlying body wall. Next, the limb bud was covered
with a filter paper (approximately 1-5 mm2) saturated with a 1:2000 solution of
colchicine (Sigma Co.). The larvae were treated for 25 min. After that period the
piece of filter paper and the tinfoil were removed and the animals returned to
Holtfreter's Solution. Most of the animals (over 90 %) survived this treatment.
The right foot remained unaffected and developed normally, thus being used as
a control for each animal. In addition, the same experimental manipulations
were repeated on control limb buds with the exception that the filter paper was
soaked in Holtfreter's Solution instead of colchicine. When the control foot had
180
P. ALBERCH AND E. A. GALE
Table 1. Digital reductions obtained by colchicine treatment
A. XENOPUS LAEVIS
Phalangeal formula
Specimen No.
X-l
X-2
X-3
X-4
X-5
X-6
X-7
X-8
X-9
X-10
X-ll
X-12
X-13
X-14
X-15
X-16
X-17
X-18
X-19
X-20
X-21
X-22
X-23
X-24
X-25
X-26
X-27
X-28
X-29
X-30
X-31
X-32
X-33
X-34
X-35
Stage at time
of treatment*
52
52
52
52
52
52
52
52
52
52
52
52
53
53
53
53
53
53
53
53
53
53
54
54
54
54
54
54
54
55
55
56
56
56
56
A
r
Experimental foot
0-2-2-4-3
0-2-2-4-3
0-2-3-4-3
0-2-3-4-3
0-0-2-3-1
0-0-Mt-4-3
1-2-3-3-3
1-2-3-4-3
0-2-2-3-0?$
0-0-3-3-071:
0-0-2-4-0?$
0-0-0-0-0$
Mt-2-3-3-2
Mt-2-3-3-2
Mt-2-3-3-2
Mt-2-3-4-3
0-2-3-3-2
0-2-3-3-2
0-2-2-3-Mt
1-2-3-3-2
1-2-3-4-3
2-2-3-4-3
Mt-l-2-3-Mt
Mt-1-2-3-1
Mt-1-2-3-2
1-1-2-3-1
1-1-2-3-1
1-2-3-3-2
0-1-2-3-2
Mt-1-2-3-1
2-2-3-3-2
1-1-2-3-1
1-1-3-3-2
1-2-3-3-2
2-2-3-4-2
Control foot
No of
elements lost
N
N
4
4
3
3
10
9
2
1
9
N
11
N
N
N
N
N
N
N
11
19
4
4
4
2
5
7
8
3
1
0
8
7
6
6
6
3
7
7
2
6
4
3
1
Nf
N
N
N
N
N
2-2-3-3-3
2-2-3-3-2
N
N
N
N
2-2-3-4-2
N
N
N
N
N
N
N
N
N
N
N
N
[15 controls - all normal]
fully developed, the animal was preserved in 10 % buffered formalin. At that
time, which corresponded to the metamorphic climax in Xenopus (N&F stage
60), all elements have undergone endochondral ossification. The axolotls, a
neotenic salamander which does not undergo metamorphosis, were allowed to
grow well beyond (about one to two months) the termination of the development
in the control limb. Consequently, at the time of preservation the axolotls were
Amphibian limb development
181
Table 1. cont.
B. AMBYSTOMA MEXICANUM
Phalangeal formula
A.
Stage at time t
'
Control foot
Specimen No. of treatment^ Experimental foot
A-l
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-ll
A-12
A-13
A-14
2D
2M>D
2M>D
2VzD
2VzD
2V2D
2V2D
2V2D
3D
3V2D
3V2D
3*/>D
3V2D
4D
1-1-3-2-0
Mt-1-3-2-0
1-1-3-2-0
l_l_3_2-0
1-2-3-2-0
1-1-2-3-2
2-2-3-3-2
2-2-3-4-2
1-2-3-2-2
1-2-2-3-2
2-2-2-3-2
2-2-2-3-2
2-2-2-3-2
2-2-2-3-1
2-2-3-3-2
2-2-3-3-2
N§
N
N
N
N
N
2-2-3-3-2
2-2-3-3-2
N
N
2-2-3-3-2
2-2-3-3-2
No. of
elements lost
7
8
7
7
6
4
1
0
3
3
2
2
2
3
[5 controls - all normal]
* Niewkoop and Faber, 1956
tNormal (i.e., 2-2-3-4-3)
X Not included in the analysis
§ N = 2-2-3-4-2
\ Developmental stage based on the number of externally visible toes (e.g. 2V^D = two welldifferentiated digits plus an early third)
undergoing endochondral ossification, with no further development occurring in
the experimental limb. To further prove this point, we allowed some Xenopus
to completely metamorphose and grow as froglets prior to preservation. The
experimental limb showed the same affected morphology as in the animals
preserved at metamorphic climax. A list of specimens treated and their developmental stage at the time of operation is given in Table 1.
Preserved animals had their soft tissue cleared with KOH after trypsin treatment and stained in toto with Alcian blue 8GX (Sigma Co.) for mucopolysaccharides (cartilage) and alizarin red S (Sigma Co.) for calcium deposits (bone)
(method slightly modified after Wassersug, 1976).
To test for any degree of cell death resulting from the colchicine treatment, as
well as its spatial distribution, two methods were used: 1) Vital staining with
neutral red (Sigma Co.). One hour after treatment, two Xenopus were placed in
a solution of neutral red (following procedure outlined by Cameron & Fallon,
1977«) for 90min followed by rinsing with dechlorinated tap water. The two
hindlimb buds of each animal were excised, observed under a microscope for
differential staining, fixed in 10 % buffered formalin and mounted on microscope
182
P. ALBERCH AND E. A. GALE
slides with Permount for further reference. The same procedure was repeated
with another two specimens 24 h after treatment. 2) Light microscopy ofhistological sections. Both hindlimb buds of four experimental animals preserved 2 and
24 h after treatment were embedded in paraplast, serially sectioned (10 /mi) and
stained with toluidine blue O (e.g. Humason, 1979). Differences in cell morphology, mitotic activity and cell death between experimental feet and opposite
control feet were studied.
To quantify differences in growth rates between the experimental and control
limbs, camera-lucida outlines of both feet of six Xenopus laevis were drawn at
the time of treatment. This was repeated every 5 to 7 days for the following 9
weeks. The area of each foot was computed each week by use of a digitizer. Thus
we obtained individual longitudinal growth curves.
Ontogenetic series of both species were prepared by preserving groups of
three axolotls at 5-day intervals and five Xenopus at 7-day intervals throughout
hindlimb development. The preserved animals were then staged and cleared and
stained by techniques referred to above.
RESULTS
Normal ontogeny
The timing of differentiation of the various metatarsal and phalangeal elements during Xenopus and Ambystoma normal limb development is shown in
Table 2. The data are based on specimens cleared in toto and stained with Alcian
blue. Therefore, we score the presence of an element only after chondrogenetic
condensation has occurred and cartilage matrix mucopolysaccharides are being
secreted. In Table 2 we report the number of elements present at every stage. For
example, at stage 53 in Xenopus, the metatarsal (MT) of only the fourth digit is
differentiated, while at stage 54+, the metatarsal of the first digit is just starting
to chondrify while digits 2 through 5 have metatarsals and digits 3 and 4 already
have differentiating first phalanges. The numbers refer to phalanges from
proximo to distal level. A dot is placed over the symbol to indicate that chondrogenesis is just beginning.
In axolotls (Table 2A), digits 1 and 2 differentiate almost in synchrony (Fig.
2A). They are sequentially followed by digit 3 (Fig. 2B), 4 and 5 (Fig. 2C). With
the exception of the first two toes, each digit differentiates before the first
phalange of the following digit starts to condense. Digit 5 is clearly the last to
differentiate; all other digits have completely differentiated before the first
phalange of digit 5 is formed.
The clearly asynchronous and anteroposterior sequential development of the
axolotl foot contrasts with the more simultaneous proximodistal differentiation
observed in Xenopus (Table 2B). At stage 52 the Xenopus limb bud is still afieldof
mesenchymal cells (Fig. 1). The metatarsals of digits 3 and 4 start to differentiate
Amphibian limb development
183
Table 2. Normal sequence of differentiation of the skeletal elements of the toes
Digits
A. Ambystoma Mexicanum
i
3D
2
2
2
2
2
Mt
Mt
2
2
2
2
2
3V2D
4D
4V2D
5D
vt
Mt
i
Mt
3§
3
3
3
3§
4§
4
Mt
Mt
Mt
Mt
Mt
i
i
i
i
Mt
Mt
Mt
Mt
i
2
1
3
3
3
3
to-
Mtt
2V2D
IV
to-
2D
III
to-
II
•CO
I
-co
Stage*
3
4
4
2
3
3
i
Mt
i
2
B. Xenopus Laevis
53
53 +
54 +
55-
i
1
56 +
to- to-
55
55 +
Mt
Mt
57
2
56
Mt
Mt
Mt
i
to- to-
5454
2
2
2
* Stages as in Table 1
t Digit number I (anterior) —> V (posterior)
$ Symbols refer to the most distal element present in each digit at the specific stage.
MT = metatarsal; 1,2,3,4 refer to phalanges from proximo to distal level
§ Phalanges appear to form by splitting of existing cartilaginous element.
at stage 53; they are soon followed by the differentiation of the metatarsals of
digits 2 and 5. Digit 1 is clearly the last digit to differentiate.
In addition, we encountered a pattern of proximodistal phalangeal differentiation in the axolotl which we did not observe in Xenopus. As indicated in Table
2A, the appearance of the phalangeal elements in digits 3 and 4 is not in simple
sequence. In these toes, a second phalange appears in the form of a continuous
cartilaginous rod that subsequently splits into two elements in digit 3, or three
in digit 4 (Fig. 2D). This exception in the sequence of proximodistal differentiation has also been pointed out by Smith (1978) in regenerating urodele limbs. In
addition, Maden (1981a) discusses the fact that the digits form before the wrist.
This same phenomenon can be observed in Fig. 2A.
Foot growth after experimental treatment
We quantitatively describe the differences in growth patterns between the
untreated control limb vs. the colchicine-treated experimental limb in Xenopus.
184
P. ALBERCH AND E. A. GALE
1
Fig. 1. Stage-52 Xenopus laevis limb bud. No chondrogenetic condensations have
occurred yet. Bar equal to 0-1 mm.
Some representative curves to show the variation in longitudinal growth encountered are illustrated in Fig. 3. While there is a marked difference in rates of
growth among individuals, all normal growth curves are characterized by a
period of approximately exponential growth during the stages of digit differentiation (53-57). On the other hand, the colchicine-treated limb bud stops
growing for a varying length of time. In most cases the period of arrestment of
absolute growth lasts several weeks at 18 °C. (More detailed measurements on
these aspects of the dynamics of growth and how they relate to differentiation are
presently being performed in our laboratory.) After this somewhat surprisingly
Fig. 2. Cleared and stained axolotl digital ontogeny: A) two-digit stage, the metatarsals of digits 1 and 2 have differentiated; B) three-digit stage; C) four-digit stage.
Note the clearly asynchronous development of the toes. In Figs C and D the peculiar
'splitting' of the cartilaginous rod to form phalanges 2 and 3 can be observed in the
fourth toe. Bar equal to 1 mm.
Amphibian limb development
Fig. 2
185
186
P.
ALBERCH AND E. A.
control
62
GALE
+
621
10
20
30
40
50
60
70
80
10
20
30 40 50 60 70
Time (days)
C
control
experimental
62 +
0 o=^
0
10
20
30 40 50
Time (days)
60
70
80
10 20 30 40 50
Time (days)
Fig. 3
60 70 80
Amphibian limb development
4A
187
B
Fig. 4. Detail of limb bud treated with colchicine (A). Cells exhibit a rounded
morphology and are more closely packed together than in the normal limb bud (B).
Also some cell death and extracellular debris can be observed. Bar equal to 0-05 mm.
long period, the experimental feet recover and begin to grow at gradually increasing rates. No catch-up growth (Williams, 1981) has been observed.
Consequently, as is shown in the figures, the treated foot at the time of termination of the developmental process is substantially smaller than the control.
Cytological effects of colchicine
Colchicine is a well-known mitotic inhibitor affecting microtubule assembly
(Hooper, 1961; Borisy & Taylor, 1967). Preliminary histological results have
shown that after colchicine treatment no mitotic activity is observed in the
treated limb bud. Cells, as early as 2h after the treatment, exhibit a rounded
morphology and are closely packed together (Fig. 4). Their morphology
contrasts with the opposite untreated limb bud (Fig. 1). The untreated limb-bud
cells show a typical mesenchymal morphology with abundant extracellular
matrix. No localized cell death was observed in the experimental limb by vital
staining with neutral red. The mesoderm stained and destained uniformly. Histological sections, however, showed some leucocytes, pycnotic dark-staining
inclusions, and extracellular debris usually associated with cell death (Fig. 4).
However, in agreement with the vital staining, we did not observe any welldefined regions of necrosis. Therefore, we tentatively conclude that, although
cell death occurred, it was not localized in any particular area of the developing
limb bud. Detailed measurements of the effect of colchicine on cell dynamics are
in progress.
Fig. 3. Longitudinal curves of foot growth of four Xenopus laevis with one treated
limb bud (diamond - growth curve for untreated limb bud; dot - growth curve for
treated limbs). Numbers on the growth curves indicate Nieuwkoop & Faber developmental stages of the respective foot. The curves correspond to the following
specimens (Table 1): A) X-21; B) X-9; C) X-5; D) X-6.
188
P. ALBERCH AND E. A. GALE
Ambystoma mexicanum (N = 14)
93%
PH4
43%
PH3
PH2
57%
PHI
7%
43%
36%
MT
Digits
Xenopus laevis (N = 35)
68%
PH4
PH3
42%
65%
PH2
90%
35%
3%
26%
PHI
58%
6%
3%
6%
MT
32%
6%
Digits
Fig. 5. Summary of the loss of skeletal elements in colchicine-treated animals. Numbers within the boxes indicate the percentage of specimens that have lost the given
element. Columns represent digit number, while rows are: MT- metatarsal; PH1-4
- phalanges one to four.
Amphibian limb development
189
Experimentally induced alterations in phalangeal number
A summary of the morphologies obtained by treating the limb bud with colchicine is presented in Table 1 and Fig. 5. Table 1 is a list of all specimens treated,
while in Fig. 5 we graphically summarize these data. Every box in Fig. 5
represents a digital element. For Xenopus, the normal phalangeal formula is:
digit 1 (metatarsal, MT, plus 2 phalanges); digit 2 (MT + 2 phalanges); digit 3
(MT + 3 phalanges); digit 4 (MT + 4 phalanges); digit 5 (MT + 3 phalanges) or
2 , 2 , 3 , 4, 3. The complete formula for the axolotl is 2,2, 3,4,2. The percentage
of treated individuals that have lost a given element is shown within the corresponding box. For example, 68 % of the experimental Xenopus (21 specimens)
lost the fourth phalange of digit 4. One axolotl and two Xenopus were unaffected, i.e., they exhibited a normal phalangeal composition in the experimental
foot, although a substantial overall size reduction was present as compared with
the control foot.
Of the 38 experimental Xenopus, there were seven feet from which phalangeal
formulas could not be reliably taken. Three of the unusable feet had fused
elements. The homologies of the digits of three feet, two with only two digits
(Nos. X-10, X-11) and one with three (No.X-9), could not be reliably ascertained
Fig. 6. Cleared and stained (A) control right foot and (B) treated left foot of
Xenopus laevis (specimen No. X-29). Note the smaller size of the treated foot.
EMB76
190
P. ALBERCH AND E. A. GALE
and were excluded from our analysis. Another specimen lost all the distal elements beyond the tibia and fibula in the treated foot (X-12). The homologies of
the toes in the remaining Xenopus with missing digits were elucidated on the
basis of presence of claws and positioning with respect to tarsal elements. A
particularly unusual specimen (X-7), used in the experimental results, had lost
the tibia and fibula while retaining all the more distal elements with the exception
of two terminal phalanges.
Fig. 5 shows two types of foot reduction in both Xenopus and Ambystoma: loss
of a whole digit and loss of individual elements. In Xenopus, digit 1 is lost much
more frequently than any other digit. In contrast, axolotls lost only digit 5. Both
Xenopus and axolotls showed digital loss in about one third of the cases. However, loss of a whole toe is not necessarily accompanied by extreme proximodistal reduction in the remaining elements. This phenomenon is illustrated in Figs
6 and 7.
Loss of individual elements also shows a different pattern in Xenopus than in
Ambystoma. In Xenopus the terminal elements of digits 1, 4 and 5 are most
frequently lost, 90 %, 68 % and 65 % of the cases respectively. The first phalange
(58 %) of digit 1, the third phalange of digit 3 (35 %), the second phalange of digit
2 (35 % ) , and the second phalange of digit 5 (26 %) are the following three most
7A
Fig. 7. Cleared and stained (A) treated left foot and (B) control right foot of Ambystoma mexicanum (specimen No. A-5). Note the smaller size of the treated foot.
Amphibian limb development
191
frequently lost elements. In axolotls, the terminal phalange of digit 4 is by far the
most frequently lost element (93 % of the cases). The terminal phalange of digit
1 (57 %) has the second highest percentage. Besides those two elements, the
axolotl foot is only repeatedly affected at three other phalanges (excluding the
loss of the whole of digit 5 (36 % of the cases)): the second phalange of digit 2
(36 %), the third phalange of digit 3 (43 %) and the third phalange of digit 4
(43%).
DISCUSSION
There has been a recent surge of interest in the connections between the
processes of growth and pattern formation during development (e.g. see Maden,
1981a; Summerbell, 1981a; Cooke & Summerbell, 1981; Cooke, 1982) such as
the dependence of mechanisms of pattern specification on the scale and dimensions of the embryonic field. We relate the results reported in this paper to this
general issue. We propose that the effect of colchicine is due to its action in
reducing the number of mesenchymal cells in the developing limb bud. This is
simply a working hypothesis consistent with our experimental results. We are
presently testing it through detailed analysis at the cellular level, using standard
histological techniques and autoradiographic labelling. We have shown that
colchicine causes the treated limb to be much smaller than the control limb (Fig.
3). The observed differences in overall size are concomitant with a reduction in
number of mesenchymal (and prechondrogenic) cells in the experimental limb
bud. This initial reduction in cell number is subsequently amplified during
development due to the properties of the exponential growth curves that characterize embryonic cell proliferation (e.g. Bertalanffy, 1960; Katz, 1980). These
changes affect subsequent processes of morphogenesis and pattern specification
resulting in varying degrees of loss of skeletal elements.
We believe that temporary arrest of mitotic division is the main role of colchicine, rather than colchicine having a more specific role, for example, through
affecting structural gene expression. Our contention is supported by reports that
other mitotic inhibitors that affect cell proliferation through very different
physiological pathways (specifically chloroethylamine (Tschumi, 1953), vinblastine (Kieny, 1975) or cytosinearabinoside (ARA-C) (Raynaud, 1981) have a
very similar effect, that is, an ordered sequence of digital reduction. In addition,
Schmalhausen (1925) showed that even malnutrition or abnormally high temperatures at critical stages during limb morphogenesis retarded the development
of the postaxial portions of the limbs of the axolotl. Presently, we are repeating
the same experiments that we report here but using ARA-C instead of colchicine. If our hypothesis is correct, we expect to obtain the same results.
Colchicine does not terminally stop differentiation events at the time of administration. Differentiation does occur after the treatment. For example, limb
buds treated at stage 53 (see results in Table 1) do not result in limbs with only
192
P. ALBERCH AND E. A. GALE
three metatarsals (the number of elements present at stage 53; from Table 2).
Our data also show that our experimental perturbations affect two processes of
pattern formation independently. These processes are: one, the determination
of the anterioposterior elements (differentiation of digits) and two, the
proximodistal differentiation of phalanges. This dissociation between the determination of the anteroposterior vs. the proximodistal axis is a fact that agrees
with current models of limb pattern formation (e.g. Hinchliffe & Johnson, 1980;
Stocum & Fallon, 1982; Tickle & Wolpert, 1981). We discuss the implications
of our results separately for each of these axes.
Anterioposterior axis. As previously pointed out by many authors on comparative (e.g. Holmgren, 1933; Saint-Aubain, 1981; Hinchliffe & Johnson, 1980)
or experimental grounds (Maden, 1981/?), there are significant differences between frogs and salamanders in the sequence of appearance of the toes. In
salamanders, at least based on the axolotl data (Table 1A), there is a welldefined anteriorposterior sequence of differentiation, as follows: Digits (1-2) —»
3—> 4—»5. Conversely, in anurans, the sequence seems to occur from the centre
towards the periphery, (3-4)—»(2-5)—> 1. In both cases, experimental reduction of the size of the limb bud via colchicine results in the loss of the element
that appears last in normal ontogeny. 36 % of the axolotls treated lost the 5th toe
(the only digit completely lost in our sample), and 32 % of the Xenopus lost their
first toe. It is not obvious how to reconcile these qualitatively different
ontogenetic patterns and results with models proposing a single organizing
centre, like the zone of polarized activity (ZPA), described in chick morphogenesis (see Summerbell & Honig (1982) for a recent review). Cameron &
Fallon (19776) report the presence of a posteriorly located region with ZPA-like
effects in Xenopus and Slack (1977) argues for the presence of an anteroposterior
polarizing region in urodele limbs.
Regardless of the mechanistic model, our data clearly show: one, the last digit
specified in ontogeny is the first to be experimentally affected; and, two, there
is no correlation between loss of a whole toe and loss of terminal phalanges. This
second point is illustrated by the fact that specimens with four toes do not
necessarily lose many phalanges on the other digits (Table 1). If digital loss
occurred by global truncation of ontogeny, one would expect the following
formulas (from Table 2): Axolotl - 2-2-3-1-X; Xenopus - X-MT-1-l-MT (X =
lost). Examination of Table 1 clearly shows this not to be the case. {Xenopus that
have lost the first toe have more phalanges in the remaining digits (Table 3).) In
fact, six out of eight experimental Xenopus have lost less than two additional
terminal phalanges and two specimens have a full complement in the remaining
four toes (Table 3). The same lack of correspondence is found in axolotls (Table
4), although in this case the difference is in the opposite direction, i.e., instead
of having more elements, they have lost more elements in the first and second
digits than would be expected. For example, Fig. 7 shows an example of a
'4-toed' Ambystoma that has lost the terminal phalanges in the first and fourth
Amphibian limb development
193
Table 3. Phalangeal formulas of experimentally induced 4-toed Xenopus Laevis
(from Table 1A)
Number of specimens
2
2
2
1
1
Phalangeal formula
0-2-3-4-3
0-2-2-4-3
0-2-3-3-2
0-1-2-3-2
0-2-2-3-Mt
No. of lost phalanges
in toes II-V
0
1
2
4
5
Table 4. Phalangeal formulas of experimentally induced 4-toed Ambystoma
Mexicanum (from Table IB)
Number of specimens
Phalangeal formula
No. of lost phalanges
in toes I-IV
1
3
1
1-2-3-2-0
1-1-3-2-0
Mt-1-3-2-0
3
4
5
toes. This independence between whole digital loss vs. distal phalange loss is also
evident in the phylogenetic data (Alberch & Gale, 1983 and in preparation).
Proximodistal axis. Two possible mechanistic interpretations can be given to
the results on the proximodistal pattern of differentiation. One, the number of
mesenchymal cells in the limb bud is reduced and chondrogenesis is dependent
on a critical minimum number of mesenchymal cells (Newman, 1977).
Therefore, as development proceeds in limbs with reduced number of cells, the
system runs out of available cells prior to the differentiation of the terminal
elements. The second would be along the lines of the 'progress zone' model
(Summerbell, Lewis & Wolpert, 1973) based on positional information. This
model argues that the fate of cohorts of cells is sequentially determined at the
apical tip of the limb where cells are actively proliferating. Cells acquire their fate
('positional value') as a function of time spent in the apical region of the limb
bud. After several cell divisions, cells are 'pushed' back relative to the apical tip
and then differentiate into a specified element. Therefore, there is a direct
relationship between number of divisions of mesenchymal cells in the apical
growth zone of the limb bud and the number of skeletal elements laid down
proximodistally (Lewis, 1975, and related X-irradiation experiments (Wolpert,
Tickle & Samford, 1979; Summerbell, 1980)). There are subtle, but conceptually
important, differences between these two hypotheses. In the first, the number of
skeletal elements is dependent on a threshold value in absolute number of
mesenchymal cells, while in the second the dependence is on number of cell
divisions.
194
P. ALBERCH AND E. A. GALE
Summerbell (1981a) has thoroughly reviewed the relationships between
growth, size and pattern in the developing chick limb bud. However, as shown
by Maden & Goodwin (1980) and Maden (19816), it is dangerous to extrapolate
from avian to amphibian development, since the systems appear to behave quite
differently regarding their regulative properties. Our results are in agreement
with most models of limb morphogenesis, where the pattern is specified by either
number of cell divisions (Summerbell et al. 1973), a monotonic gradient (e.g.
Tickle, Summerbell & Wolpert, 1975; Summerbell, 19816; Cooke & Summerbell, 1981) or a more complicated diffusion-reaction model that generates a prepattern of regions of various levels of morphogen concentration (e.g. Wilby &
Ede, 1975; Newman & Frisch, 1979; Meinhardt, 1982). Without intention to
enter into a discussion of the pros and cons of each of the models proposed by
these authors, it suffices to say that they are all to some degree boundary dependent, i.e., a change in the size of the domain of interaction can result in a
qualitative change in global pattern. In addition, there are comparative studies
on genetic mutants that illustrate that changes in number of digits are correlated
with changes in the dimensions of the early limb bud, for example, the oligosyndactyly (Os) mutation in mice (Griineberg, 1963). Similarly, work with
teratogens (e.g. the work by Messerle & Webster (1982) on cadmium-treated
limbs) shows that limb defects associated with digital loss are correlated with
limb primordia of reduced dimensions. In addition, Sewertzoff (1931), Raynaud,
Gasc & Renous-Lacuru (1974) Raynaud et al. (1975) and Raynaud (1976) have
shown that in species of lizards characterized by digital loss the limb bud is
comparatively smaller, a fact that Raynaud (1976) has argued is due to a reduction in the migration of mesodermal cells into the limb bud primordia.
Given this perspective, which stresses the importance of scale in limb morphogenesis , it is not surprising that one of the best documented roles of the two major
organizing centres in limb morphogenesis, the AER and the ZPA, is to regulate
mitotic activity (see Summerbell, 19816, for review). Furthermore, if our contention is correct, one would expect a fair degree of invariance in the absolute
size of the limb bud at the time of pattern specification among species sharing the
same limb skeletal pattern regardless of the final size of the organism. This
hypothesis is not in agreement with Maden (1981a), who conclusively showed
that urodele limb regeneration is size independent. One would have to argue that
there are differences in this respect between limb regeneration and normal
development. This would not be surprising, since the problem of size dependence vs. independence is probably a quantitative rather than qualitative issue.
Some developmental systems are more stable to perturbations in boundary conditions than others. As Maden (19816) has shown, the concepts of mosaic vs.
regulative development are part of a continuum which varies ontogenetically and
phylogenetically.
In conclusion, we have reconfirmed the qualitative differences between the
ontogenies of anurans and urodeles by direct examination and by studying their
Amphibian
limb development
195
response to the same experimental stimulus. Our results also suggest that pattern
specification in the amphibian limb is dependent on either number of undifferentiated cells or absolute dimensions of the limb bud, and that pattern specification
mechanisms along the anterior-posterior and proximodistal axes are independent to some degree.
We would like to thank the Axolotl Colony, University of Indiana, for sending the Ambystoma mexicanum embryos. Anna Haynes and M. Maden critically read the manuscript and
offered useful comments. We thank Laszlo Meszoly for the illustrations and A. Coleman for
the photographic work. Catherine McGeary provided assistance in thefinalpreparation of the
manuscript. The work was supported by NSF Grant DEB-81-20917.
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{Accepted 1 April 1983)