J. Embryol. exp. Morph. Vol. 70, pp. 241-260, 1982
Printed in Great Britain © Company of Biologists Limited 1982
241
Protein synthesis during limb regeneration
in the axolotl
By J. M. W. SLACK 1
From Imperial Cancer Research Fund, Mill Hill Laboratories, London
SUMMARY
A study has been made of limb regeneration in the axolotl using two-dimensional gel
electrophoresis of proteins labelled with [35S]methionine.
In the early stages of regeneration seven proteins are identified which are-specific to the
mesenchyme and thirteen which are specific to the epidermis. There is very little change in
the gel pattern until the onset of overt cytodifferentiation upon which the muscle and cartilage
become substantially different both from each other and from the blastemal mesenchyme.
The gel pattern of mesenchyme from the larval limb bud is almost identical to that of the
blastemal mesenchyme, while the pattern of the limb-bud epidermis differs somewhat from
the blastemal epidermis.
A careful search has been made for differences which might be associated with 'positional
information' by comparing forelimb with hindlimb, proximal with distal and anterior with
posterior. The differences which have been found are all associated with differences in visible
cellular composition or in rates of differentiation, rather than with position per se. It is concluded that positional codings cannot be detected by this technique. On the basis of biological
experiments, six criteria are proposed by which to assess future searches for positional codings.
INTRODUCTION
This paper is concerned with a descriptive biochemical study of limb regeneration. The method used was to study the pattern of protein synthesis at different
stages of regeneration and in different regions of the blastema using highresolution two-dimensional gels. Since several hundred protein species can be
resolved, this technique enables more substances to be examined simultaneously
than any other single biochemical technique. It can thus give us the best measure
available of the overall 'state' of the constituent cells, which may be defined in
the present context as a list of which genes are active and how active they are.
There were four distinct reasons for undertaking the work. The first object
was to discover major tissue-specific proteins which could serve as markers of
the differentiated cell types. This is necessary for in vitro studies of limb development and regeneration, both for quantification of differentiation and for confirmation of the identification of cell types made histologically.
The second object was to find out how much biochemical change occurs prior
1
Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories,Burtonhole
Lane, London, NW7 IAD, U.K.
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J.M.W. SLACK
to overt cytodifferentiation of the blastema in the so-called 'morphogenetic'
phase of development. Both the epidermis and the mesenchyme were studied.
The third object was to compare the regeneration blastema with the larval
limb bud. Discussion has raged for many years over whether regeneration is or
is not like embryonic development (e.g. Faber, 1971), but this type of study
makes possible a clear answer by comparing the spectrum of proteins synthesized by the blastema and the limb bud.
The fourth object was to find biochemical correlates for 'non-equivalence', or
the codings which are presumed to control pattern formation in the blastema.
The criterion adopted was to look for differences between tissues of the same
cell type from different positions within the blastema.
The results enable clear decisions to be made about the first three but not
the fourth of these questions. The failure to find a biochemical basis for nonequivalence is considered further in the Discussion.
METHODS
Preparation of samples
The blastemas were obtained from axolotls of about 8-12 cm in length which
were reared individually from eggs in the laboratory. At this age and at the
temperature of the aquarium the cone stage of regeneration was reached about
two weeks after amputation, the palette stage by three weeks and the digit stage
by four weeks. These stages correspond approximately to Iten & Bryent's (1973)
' medium bud', 'late bud' and 'early digits' stages, and sections of the blastemas
used are shown in the Results. Larval limb buds were obtained from animals
about 1-5 cm in length and dermis from young adults about 16-18 cm in length.
The blastemas were removed from the animals with iridectomy scissors and
dissected in NAM ('normal amphibian medium'- Mohun, Tilly, Mohun &
Slack, 1980) containing 20 mM glucose. The epidermis was separated from the
mesenchyme without the use of trypsin and the other dissections performed are
depicted in Fig. 1. Similar fragments from 3-6 blastemas were pooled, cut into
small pieces, and put in Eppendorf tubes. They were rinsed in the labelling
medium (without label) and then incubated for 6 h at 25 °C in 50 (A. of medium
containing 30-80 /iC\ of [35S]methionine (> 600 Ci/mmole Amersham) or
100/*Ci of [32P]orthophosphate (carrier-free, Amersham). The medium was
70% Eagle's, respectively methionine or phosphate-free, with antibiotics in the
concentrations used for primary tissue culture from amphibian material (Laskey,
1970). No serum was included. The tubes were kept in a gas-tight box filled with
5 % CO 2 :95% air. Under these conditions incorporation of ^S into acidinsoluble material was linear over 18 h, but the shorter labelling period of 6 h
was chosen in order to exclude all possibility of artifacts due to bacterial contamination. All the experiments involved this labelling period except for the
adult dermis which was labelled for the full 18 h.
Foreign synthesis during limb regeneration
Ant.
Mid.
243
Post.
-Apical cap
Dense mesenchyme
Loose mesenchyme
Epidermis
Digits
IV
Post.
Ant.
'Muscle'
Skin
Cartilage
Fig. 1. (a) A palette-stage blastema showing dissection into anterior, middle and
posterior parts, (b) A digit-stage blastema showing dissection of the digits and the
regions of differentiating tissues on the proximal surface.
After incubation the samples were rinsed twice in NAM/glucose containing
1 mM unlabelled methionine and then frozen in dry ice. They were not stored
in this state but were processed immediately by a method essentially similar to
that of Garrels (1979). Each sample was thawed and homogenized at 4 °C in
200/d of micrococcal nuclease solution (50/^g/ml Worthington NFCP in
2 mM-CaCl2, 20 mM Tris pH 88), then rehomogenized after the addition of
20 fi\ 5 % SDS 15% mercaptoethanol and 25 /i\ nucleases (DNAase 1 Worthington DPFF 1 mg/ml, RNAse A, Worthington RASE 0-5 mg/ml, 50 mM-MgCl2,
0-5 M Tris pH 70).
The homogenization was continued until a clear solution was obtained. This
was allowed to stand for 5 min in ice and was then frozen in dry ice and lyophilized overnight. The residue was dissolved in 100/d sample buffer and this was
244
J.M.W. SLACK
centrifuged for 2 min in a microfuge. The supernatant was transferred to
another tube, an aliquot was removed for counting by the method of Mans &
Novelli (1961) and the remainder was stored at -70°. The pellets were very
small, and when examined in the microscope proved to consist mainly of melanin
granules.
Gel electrophoresis
This was carried out essentially by the method of O'Farrell (1975). The sample
buffer consisted of 9-5 M urea, 4% NP-40, 2% ampholyte and 100 mM-DTT.
The ampholyte was LKB ampholine and was a 2:1 mixture of pH 5-7 range
and pH 3-5-10 range. Isoelectric focusing was carried out for 19 h at 500 V. No
prefocus was used, instead the power pack was set to a maximum current of
0-25 mA/gel and the voltage automatically increased to 500 V over the first 2 h
The focusing gels were 0-25 cm in diameter and 16 cm in length.
The second-dimension slab gels measured 17 x 25 cm and were uniform 10%
acrylamide gels. A 3 cm stacking gel of 2-5% acrylamide was poured on top
of this. The IEF gels were equilibrated for 30 min in 2-3% SDS, 10% glycerol,
62 mM Tris pH 6-8, 10 mM-DTT and then glued to the top of the slab gel with
1 % agarose made up in the same buffer. Electrophoresis was for 16 h at
20 mA/gel. For many of the gels 14C-labelled molecular weight markers
(Amersham) were run in slots at both ends of the focusing gel.
The slab gels were fixed in 25% methanol, 15% acetic acid for 1 h, then
processed for fluorography by the method of Bonner & Laskey (1974). In most
cases 0-5xl0 6 cpm were loaded and a suitable image was obtained on the
Fuji X-ray film after 1 week at — 70 °C. 32P-labelled gels were visualized either
by autoradiography or with the aid of an intensifying screen behind the film.
Histology
Blastemas and limb buds were fixed for 4 h in 2-5% glutaraldehyde in 0-2 M
phosphate buffer, pH 7-4. They were dehydrated, embedded in Araldite, and
sectioned at 1 fim. The sections were stained with 1 % toluidine blue in 1 %
borax.
Reproducibility of "2-D gels
The gels were compared with one another in two ways. They were judged to
be qualitatively identical when no spot present on one was completely absent
from the other, and they were compared quantitatively by ringing the 150 most
prominent spots on each gel and asking how many were the same ones. Neither
of these methods is entirely satisfactory. The qualitative comparison will involve
more spots the longer the gels are exposed to the X-ray fim, and clearly if 700
of 700 spots are identical this is a more stringent test of similarity than if only
200 of 200 are. The quantitative assessment depends on a subjective assessment
of which spots are most 'prominent'. But this is unavoidable without a 2-D
Protein synthesis during limb regeneration
245
densitometric scanner, and such machines are expensive and require elaborate
computer facilities to interpret the data (Bossinger et al. 1979).
When the same sample was run twice the gels were qualitatively identical and
the 'quantitative similarity' was about 75%. When a blastema was dissected
into epidermis and mesenchyme the gel of the complete blastema contained all
the spots found on the gels of the separate tissue (see Fig. 3). When a similar
sample was prepared from different animals there could be a few minor spots
different, possibly due to genetic variation or to slight differences in sample
preparation. Altogether animals from seven spawnings were used in these
experiments, and although the use of outbred animals should not affect the
main biochemical conclusions, it is very important to repeat experiments where
the significance of small differences is in question.
Altogether, the experiments described here involved the running of 125 slab
gels.
RESULTS
Normal regeneration of the forelimb
All of the experiments discussed in this section were carried out on regenerates from amputations through the mid-upper forelimb. In Fig. 2a is shown a
horizontal section of a cone-stage blastema similar to those used in the experiments. At this stage the internal tissue is very uniform and consists of approximately 90% blastemal mesenchyme cells, 4 % macrophages, 3 % capillary
endothelial cells and 3 % erythrocytes. The mitotic index is about 1*5%. The cell
types are shown in Fig. Id. The epidermis consists of about 96 % epidermal cells
which show some stratification from inside to outside, 3 % Leydig gland cells
and 1 % macrophages (Fig. 2c). There is no obvious regional variation except
for the apical cap, in which there are about 10% macrophages at this stage.
The cell counts indicate that the gel pattern for' epidermis' and 'mesenchyme'
overwhelmingly reflects the activities of the epidermal cell and the blastema
mesenchyma cell respectively. The minority cell types should not be forgotten,
however, since a major protein specific to one of them might be visible as a
minor spot in the overall pattern.
Two-dimensional gels of whole-cone-stage blastema, cone-stage mesenchyme
and cone-stage epidermis are shown in Fig. 3a-c. All the spots present on the
gels of the separated tissues are also present on the gel of the whole blastema.
The spot marked /? is /?-actin, which was identified by peptide mapping in a
previous paper (Mohun et al. 1980). This is present on all the gels and serves
as a readily visible reference point.
The epidermal and mesenchymal patterns share many features (quantitative
similarity 57%) but there are a number of significant differences. Seven proteins
are indicated which are reproducibly found only in the mesenchyme, and
thirteen which are reproducibly found only in the epidermis. The mesenchymal
proteins are also all found in distal forelimb and in hindlimb mesenchyme. The
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J. M. W. SLACK
Fig. 2. Histology of blastemas at the stages used, (a) Cone stage: horizontal section;
ac apical cap, m mesenchyme, e epidermis. Scale bar: 0-5 mm. (b) Palette stage:
horizontal section; lm loose mesenchyme, dm dense mesenchyme. Scale bar:
0-5 mm. (c) Epidermis of cone stage showing epidermal and gland cells. Scale bar:
30 /tin. (d) Mesenchyme of cone stage showing minority cell types: mac. macrophage,
RBC red blood cell, end. capillary endothelial cell. Scale bar: 30/*m.
30 —
46 —
67 —
92—
200 —
Fig. 3. Two-dimensional gels of the cone-stage blastema, (a) Whole blastema; (b) mesenchyme; (c) epidermis. Seven proteins
specific to the mesenchyme and thirteen proteins specific to the epidermis are shown. Numbers at the side indicate molecular
weights in Kilodaltons. The pH gradient in this and subsequent gels runs from about 5-0 on the right to 7-5 on the left, ft is /?-actin.
4
SI
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248
J. M. W. SLACK
200
30 —
Fig. 4. Two-dimensional gels of palette-stage blastema, (a) Mesenchyme; the three
spots indicated appear between cone and palette stage, (b) Epidermis; the two spots
indicated increase in intensity from the cone stage.
epidermal proteins are found in epidermis from all types of blastema and also
a larval limb-bud epidermis and mature skin. All of the epidermal proteins
except 7, 8 and 13 become heavily labelled with [32P]orthophosphate, so are
presumably modified by covalent attachment of phosphate or nucleotides.
In Fig. 2b is shown a horizontal section through a palette-stage blastema
similar to those used in the experiments. The epidermis looks the same as that
of the cone stage except that there are no macrophages and there is perhaps a
slight reduction in the proportion of gland cells. The mesenchyme is no longer
uniform; in the proximal part of the blastema it forms a central condensation
surrounded by a more rarefied periphery, and a number of nerve fibres are
Fig. 5. Histology of the digit-stage regenerate, (a) Horizontal section shows the
disposition of cartilage condensations. Note that the differentiation of digit I (on
the left) is more advanced than that of digit IV (on the right). Scale bar 0-5 mm.
(b) Muscle from proximal region. Scale bar 30 /im. (c) Cartilage from proximal
region. Scale bar 30 /*m. (d) Skin from proximal region. The histology of the epidermis is the same as in the early blastema but there is now a collagen layer and
some fibroblasts making up the dermis: col. collagen, fib. fibroblasts, g gland cell.
Scale bar 30 fim.
Protein synthesis during limb regeneration
X
249
250
J.M.W. SLACK
evident, particularly in the proximal region. In the distal region the macrophages
have gone and a few melanocytes have appeared. Because of the regionalization
of the blastema it is difficult to count cells as accurately as in the cone stage, but
the distal part was composed of 96% mesenchymal cells, 2 % Schwann cells,
1 % melanocytes and 1 % endothelial cells.
The 2-D gels for the palette-stage epidermis and mesenchyme are shown in
Fig. 4. The most noteworthy feature is their close similarity to the patterns
found at the cone stage one week earlier. In the mesenchyme the three basic
proteins indicated on the figure are the only consistent differences. In the epidermis two of the low-molecular-weight spots increase greatly in relative intensity, but they are not altogether absent from the cone stage. The quantitative
similarity of the two stages is 76% for the mesenchyme and 77% for the epidermis, and this is not significantly different from the figure obtained when the
same sample is run on two different gels.
It seems therefore that there is very little development of either tissue between
cone and palette stages at least with respect to the proteins visualized on these
gels.
Figure 5 shows a horizontal section through a digit-stage regenerate, together
with high-power views of the muscle, cartilage and skin from the upper arm
region. By this stage the cytodifferentiation of the internal tissues is quite
evident: the muscle is composed of multinucleate myotubes with cross-striations,
and the cartilage cells are becoming flattened and beginning to secrete metachromatic matrix material. In the skin the dermis has begun to develop proximally in the form of a thick layer of oriented collagen fibres associated with a
small number of fibroblastic cells.
In Fig. 6 are shown gels which reflect the activities of muscle, cartilage and
skin. The samples were obtained by dissection of the upper-arm portions of
digit-stage blastema into a central region which is pure cartilage (the distal
humerus), the skin which is mainly epidermis but also includes the dermal
collagen layer and some fibroblasts, and the intermediate tissue which is mainly
muscle but contains also some nerves, blood vessels and connective tissue (see
Fig. 1).
The muscle pattern resembles the blastema mesenchyme more than the
cartilage (61 % similarity as against 40%) but both are clearly different from
the palette mesenchyme and from each other. The cartilage pattern is dominated
by a very intense spot of about 35000 molecular weight marked ' A ' on Fig. 6 b,
and by a cluster of spots around 80000-90000 molecular weight marked ' B ' .
Five of the seven mesenchymal proteins are retained.
Although the muscle pattern differs in several qualitative respects from the
blastema mesenchyme there are no super-abundant species which stand out
clearly. In fact the most prominent feature is the a-actin (identified by peptide
mapping in a previous study, Mohun et al. 1980). This protein is usually regarded
as muscle-specific, but it will be evident from the gels published here that it is
30 —
46 —
67 —
92 —
200 —
*0
B
.ft
Q
30 —
0
Fig. 6. Two-dimensional gels of differentiated tissues from the digit-stage regenerate, (a) Muscle, ' a ' is a-actin; mesenchyme-specific
proteins indicated; ' d ' shows two proteins found in distal but not proximal blastemas (see 'Non-equivalence'), (b) Cartilage. A
and B are specific features. Mesenchymal proteins 1, 3, 4, 5 and 6 shown, (c) Skin. Epidermal proteins 1-13 shown.
••
K)
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J. M. W. SLACK
present in the blastema mesenchyme to some extent and there is even a trace in
the epidermis. All seven of the mesenchymal proteins are retained.
The adult dermis may look less 'differentiated' than muscle and cartilage but
it is not particularly similar to the blastema mesenchyme (similarity 47%), so
even if much of the blastema is derived from this tissue it must undergo some
considerable biochemical changes on dedifferentiation, as do the other tissues.
The skin from the digit-stage regenerate has some obvious affinities with the
epidermis from earlier stages, for example it shows the 13 marker proteins. Its
overall similarity to palette epidermis is 57 %, but it is not clear to what extent
the differentiation has occurred in the epidermis and to what extent the change
is attributable to the presence of the dermis, which is presumably derived from
the blastema mesenchyme.
In general the conclusions from this part of the work are that the main biochemical changes are those associated with visible cytodifferentiation. During
the phase of blastemal proliferation there is little or no development in terms of
protein synthesis pattern in either the epidermis or mesenchyme.
The larval limb bud
A number of axolotl larval limb buds with a shape roughly corresponding to
the palette-stage blastema were dissected into epidermis and mesenchyme, labelled
and the proteins fractionated on 2-D gels. The appearance of the buds is shown
in Fig. 7. The mesenchyme is undifferentiated and not unlike the blastemal
mesenchyme in appearance, except that there are no macrophages. The epidermis by contrast differs from the blastema epidermis in that it is only two
cells thick and lacks gland cells.
The gels are shown in Fig. 8. There are a few minor differences between the
mesenchyme and the blastema mesenchyme but on the whole it is remarkable
how similar they are. There are only four differences from the palette mesenchyme, one of which is the absence of mesenchymal protein no. 7, and the
quantitative similarity is 70%. By contrast the epidermal pattern differs
significantly from that of the blastema epidermis (quantitative similarity 54 %),
which is not unexpected since it is clearly different in microscopic appearance.
It seems therefore that it is legitimate to regard the blastema mesenchyme
cell as having reverted to an 'embryonic' condition, since its state in terms of the
pattern of protein synthesis is very similar to the larval one.
Non-equivalence
Comparisons were made between anterior and posterior regions of the
blastema at various stages, between proximal and distal blastemas and between
fore- and hindlimb blastemas. The pairs of gels were examined for qualitative
differences which might be associated with the codings believed to control the
pattern of differentiation. The results are summarized in Table 1. No attempt
was made to compare the gels quantitatively since reruns of the same sample
Protein synthesis during limb regeneration
253
Fig. 7. Histology of the larval limb bud. (a) Horizontal section to show disposition
of epidermis and mesenchyme (apical cap is out of the plane of section). Scale bar
0-1 mm. (b) High-power view. The mesenchyme cells resemble that of the blastema
while the epidermis is much thinner and lacks gland cells. RBC red blood cell,
n nerve sheath, cap. capillary, epi. epidermis. Scale bar 30 pm.
are not identical in this respect and the method is subjective in any case. It
should be borne in mind that whereas the comparisons in the previous sections
involve prominent spots those in the present section involve minor spots. There
is a slight variability in the minor spots when experiments are repeated on
different batches of animals, presumably both because of genetic variation and
because of slight differences in the labelling and extraction procedures. This
means that the negative results are the most significant because there is no doubt
about two gels being the same. If a slight difference is found between tissue from
two positions it is essential to repeat the experiment two or three times to ensure
that it is reproducible. No great significance should therefore be attached to the
'yes' entries of Table 1 referring to single experiments.
No difference could be detected between fore- and hindlimb blastemas either
in the epidermis or the mesenchyme (Fig. 9, compare with Fig. 2). No difference
could be detected between anterior and posterior regions at either cone or
palette stage. The gels shown on Fig. 10 are from anterior, middle and posterior
regions of the palette stage and are overexposed by a factor of 3 compared with
9
EMB 7O
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J. M. W. SLACK
200 —
30 —
Fig. 8. Two-dimensional gels of larval limb-bud tissues, (a) Mesenchyme. Two
proteins are arrowed which are not found in the blastema and two spaces are
indicated in which blastemal mesenchyme proteins would be found. The boxed
spots are mesenchyme-specific proteins shared with the blastema, (b) Epidermis.
The boxed and arrowed spots are epidermis-specific proteins shared with the
blastema. There are also many differences from the blastemal epidermis.
the others. The overexposure brings about 700 spots into view, and among these
there is no difference between anterior and posterior.
In the cases where differences can be detected there is some reason to think
that they are associated not with codings but with more trivial factors such as
differences of cellular composition or differences in degree of maturation between regions. So, for example, there are three quantitative differences between
the palette-stage middle portion and the anterior/posterior portions (Fig. 11).
However, the middle portion differs in composition from the others since it
contains the apical ectodermal cap and more of the proximal mesenchymal
condensation (Fig. 1), and a few differences are therefore to be expected.
There are two spots reproducibly present in mesenchyme from blastemas
formed on distal amputation surfaces which are absent from proximal blastema
mesenchyme. However, they are both present in proximal tissue at the digit
stage (see Fig. 6a) and therefore presumably reflect the fact that a 2-week distal
255
Foreign synthesis during limb regeneration
Table 1. Regional comparisons
Level of
amputation
Stage
Forelimb
and hindlimb
Forelimb
Proximal
Cone
Proximal
and distal
Cone
Forelimb
Proximal
Cone
Forelimb
Proximal
Palette
Limb
Forelimb
Proximal
Digits
Hindlimb
Forelimb
Proximal
Proximal
Digits
Digits
Forelimb
and hindlimb
Adult
Comparisons
Forelimb vs. hindlimb
(mesenchyme and
epidermis)
Proximal vs. Distal
(mesenchymal and
epidermis)
Anterior vs. posterior
(mesenchyme and
epidermis)
Anterior and posterior
vs. middle
Anterior vs. posterior
Anterior vs. posterior
(32P label)
Digit I vs. II
III
IV
Digit I vs. IV+ V
Anterior vs. posterior
proximal region
Anterior vs. posterior
dermis from lower limb
Number of
experiments
using different animals
Differences
2
No
2
Yes1
2
No
1
Yes2
1
2
No
No
3
Yes1
1
1
Yes3
Yes3
1
Yes3
2
Samples of slightly
Reproducible differences associated with cell differentiation.
3
These differences are not the same as those found in other
different cellular composition.
A-P comparisons.
1
blastema is slightly advanced in differentiation compared to a 2-week proximal
blastema.
The four digits were compared several times and the only reproducible difference was an anteroposterior gradient of the cartilage-specific protein ' A '
(Fig. 6 b). This must be associated with the fact that there is an anteroposterior
gradient in the extent of cartilage differentiation which is clearly visible through
the microscope (Fig. 5 a). The hindlimb digits were also compared. They also
show an earlier chondriflcation in the anterior, but at the stage from which the
samples were prepared there was evidently not sufficient synthesis of the
A protein to be visible on either gel. The three minor differences between hindlimb digits I and IV + V were not the same as differences found in any experiment between forelimb digits.
The last comparisons on the table also show minor differences between
9-2
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J. M. W. SLACK
200
Fig. 9. Two-dimensional gels of cone-stage hindlimb blastemas, (a) Mesenchyme.
Boxed spots are mesenchyme-specific, found also in forelimb. (b) Epidermis. Boxed
and arrowed spots are epidermis-specific, found also in forelimb.
anterior and posterior but they are not the same differences, and since we are
now dealing with tissues possessing substantial innervation and blood supply it
is not likely that the samples compared are of identical cellular composition.
None of the positive results from this study satisfies the criteria for association
with positional codings which are outlined in the Discussion. The negative
results are most striking, and we can say with confidence that at the early stages
of regeneration no difference can be found between anterior and posterior, or
between forelimb and hindlimb, using this technique.
DISCUSSION
Several prominent marker proteins have been uncovered which are characteristic of the various differentiated cell types. These should prove useful for in
vitro studies involving cell characterization since they are likely to show up on
one-dimensional gels which separate by molecular weight alone. To establish
their identities unambiguously will require further work. However, it seems
probable that the epidermal proteins may all be keratins since their molecular
weights are approximately 65, 63, 59, 55, 50 and 39 kilodaltons (means of three
separate determinations) which gives a reasonable match to human keratins
30 —
46 —
200 —
Fig. 10. Two-dimensional gels of (a) anterior, (b) middle and (c) posterior regions of the palette-stage forelimb blastema. These gels
are exposed for three times as long as the others and show more spots as a result. Three quantitative differences are indicated
between (b) and the other two.
V
..
is
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J.M.W. SLACK
(65, 63, 55, 49, and 40 - Fuchs & Green, 1978). It was thought that the cartilage
' A ' protein might be the low-molecular-weight link glycoprotein of the proteoglycan matrix (Heinegard & Hascall, 1974). However, it did not coelectrophoresize with a sample of porcine material kindly donated by Dr T. Hardingham of
the Kennedy Institute of Rheumatology, London. It will require a functional
test to establish whether it is none the less a homologous protein.
The discovery that the larval limb-bud mesenchyme is very similar to the
blastema mesenchyme makes it reasonable to use cell lines derived from larval
buds in research on regeneration. The 2-D gel pattern provides a discriminating
test of cell affinity and should make it possible to prove that cells have not
grown from a minority type such as capillary endothelium, and have not evolved
too much during culture. Axoloti cells have so far proved refractory to tissue
culture, but other amphibian species capable of regeneration are more amenable
(Reese, Yamada & Moret, 1976).
In general, this study has shown a close correspondence between the visible
appearance of tissues and their 2-D gel patterns. If there are events occurring
during the 'morphogenetic phase' of regeneration, for example between cone
and palette stages, then they are not readily detectable.
When we consider the problem of non-equivalence it is important to decide,
in advance of doing the experiments, what criteria should be used for regarding
a biochemical difference as significant. Amphibian limb regeneration should
provide a very favourable case for searching for non-equivalence because the
extensive biological research has provided us with the data to lay down several
criteria, particularly with respect to comparisons between proximal and distal
(PD) or anterior and posterior (AP). These criteria will of course apply to any
biochemical comparison and not just to the technique employed in the present
study.
1. The comparison must be made between tissues having the same compositions in terms of visible cell type, but taken from different positions.
2. PD and AP differences should be the same in the hindlimb as in the forelimb.
3. PD differences should be shown by blastemas at all stages, and AP differences should be present in the blastema from at least the palette stage.
4. Differences should be located in the mesenchyme and its derivatives rather
than in the epidermis.
5. The same differences should be found in different individual animals and
in other species capable of regeneration.
6. The same differences should be found in larval limb buds.
Number 1 comes from the basic definition of non-equivalence (Lewis &
Wolpert, 1976). No. 2 is to be expected from morphological homology, from
fact that intercalary regeneration can be provoked between fore- and hindlimb
combination (Stocum, 1980), and from the fact that hindlimb posterior skin
can cause duplicated regenerates after transplantation to the anterior fore-
Protein synthesis during limb regeneration
259
limb (C. Dinsmore, personal communication). No. 3 derives from the autonomous differentiation of blastemas grafted to neutral sites (Stocum, 1968; de
Both, 1970), and from the estimates of AP determination time from rotation
experiments (Iten & Bryant, 1975; Tank, 1977). No 4 comes from Carlson's
(1975) finding that muscle and dermis were most effective at disrupting the
pattern of the regenerate after positional dislocation. No. 5 is to be expected if
the mechanism of pattern formation is the same in similar organisms. No. 6
comes from the fact that larval buds show similar responses to grafting experiments as do blastemas (Maden, 1981).
None of the PD or AP differences found in this study even approaches the
satisfaction of all these conditions, and they are accordingly not regarded as
significant.
What, then is the reason for the failure to find a biochemical basis for nonequivalence? One possibility is that there is no such thing as non-equivalence:
cells have no characteristic other than those associated with histological type
and the mechanism of pattern formation is radically different from the' positional
information' mechanisms proposed by many authors. This seems unlikely,
mainly because it is virtually impossible to make any sense of grafting experiments on limb regeneration except in terms of a 'second anatomy' of positional
codings (Slack, 1981).
It seems more probable that the biochemical differences involved cannot be
picked up by the technique used. The extraction procedure will leave behind
very hydrophobic proteins and the separation will not resolve proteins of very
acidic or basic isoelectric point or of very high or very low molecular weight.
Furthermore, the coding molecules may not be proteins. It seems unlikely that
proteins should not be involved at some level in the coding mechanism, since
even if the coding molecules themselves are, say, carbohydrates, there will still
be a regional difference in the enzymes which make them. However, if proteins
are involved only in a catalytic or regulatory capacity, rather than a structural
one, it is quite possible that the species concerned will be unsufficiently abundant
to show up on a 2-D gel. Current estimates of the number of genes which are
active in a given cell type suggest about 103 mRNA species of moderate abundance and about 104 mRNA species of low abundance (1-15 copies per cell)
(Davidson & Britten, 1979). Since 2-D gels can resolve at most 103 proteins it
is clear that they are looking only at the proteins formed from the moderately
abundant messages.
Even if the coding substances are sufficiently abundant to be detected we have
no guarantee at present that they are not carried by a minority subset of the
blastemal cells. If this is so then it is likely that further progress in this field will
depend on the ability to grow limb cells in vitro under conditions in which they
retain their morphogenetic properties.
I am grateful to Rita Tilly and James Vernon for the histological work and to David Trevan
and Shirley Williams for photography.
260
J.M.W. SLACK
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{Received 20 January 1981)