/. Embryo], exp. Morph. Vol. 37, pp. 33-48, 1977
Printed in Great Britain
33
The pattern of cell division during growth of the
blastema of regenerating newt forelimbs
By A. R. SMITH 1 AND A. M. CRAWLEY 2
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School, London
SUMMARY
Pulse and continuous labelling with tritiated thymidine are used for a quantitative study of
cell division rates in regeneration blastemas. Proliferation is initially uniform; later a proximodistal gradient develops in the mesenchyme, with the highest labelling index at the tip,
where practically all cells are shown to be dividing. In the ectoderm there appear to be two
growth bands, one close to the stump and the other close to the tip. The results are consistent
with the progress zone theory, and agree well with the numerical estimates of growth rates
used in our previously reported simulation.
INTRODUCTION
In a previous paper we presented quantitative data on the growth of the
blastema of the regenerating newt forelimb (Smith, Lewis, Crawley & Wolpert,
1974). A model for limb regeneration was put forward making use of the progress zone concept (Summerbell, Lewis & Wolpert, 1973). This suggests that
there is a region at the tip of the developing or regenerating limb in which cells
autonomously change their positional value with time, so as to take on a progressively more distal intrinsic character. In amphibian limb regeneration, as
viewed here, a progress zone must be set up in the blastema; the positional
value of the cells in the progress zone will initially correspond to the positional
value of the cells at the level of the cut. Our formal quantitative model provided
a good description of the observed growth curves.
We distinguish three phases of limb regeneration: the formation of a blastema
and progress zone; the laying down of the skeletal rudiments; and the subsequent slower growth of these rudiments. Here we will present data on the
pattern of cell division during the end of the first phase and the second phase,
together with some relevant histological observations. A knowledge of the
pattern of cell division is necessary for quantitative models; we were particularly
1
Author's address: MRC Demyelinating Diseases Unit, Newcastle General Hospital,
Newcastle upon Tyne NE4 6BE.
2
Author's address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London W1P 6DB, U.K.
34
A. R. SMITH AND A. M. CRAWLEY
interested to know whether all the cells in the region of the postulated progress
zone were dividing, whether cells divided less outside the progress zone, and
whether there was a difference between cartilaginous and other regions. There
is quite an extensive literature on the pattern of cell division during regeneration
(Litwiller, 1939; Chalkley, 1954, 1956; Hearson in Thornton, 1968). These
studies used mitotic index and provide almost the same type of information as
do measurements of pulse-labelling index; that is, they give estimates of the
relative rates at which new cells are formed through cell division in various
parts of the blastema at successive stages in regeneration. The use of mitotic
index as an indicator of growth, however, suffers from two limitations. First, in
the newt limb mitoses are few and far between, because the cell cycle is long
compared with the duration of mitosis. Thus it is excessively laborious to
accumulate large counts and reduce random errors to an acceptable level.
Secondly, from the mitotic index alone one cannot, for example, distinguish a
population of cells which are all dividing at the same slow rate from a population
in which some cells do not divide at all while others divide rapidly. Because the
S-phase is much longer than the mitotic phase, pulse-labelling with tritiated
thymidine gives bigger counts and smaller random errors than does the mitoticindex method. Furthermore, from continuous labelling measurements one can
gauge what proportion of the cell population is quiescent, and what proportion
actively dividing. Pulse-labelling was used by Hay & Fischman (I960, 1961) but
they made no detailed counts of the labelling index in the mesoderm. Grillo
(1971) has tried to measure the cell cycle parameters in regenerating limbs by
counting labelled mitoses. But his data, which he describes as 'preliminary',
relate chiefly to the small cone stage and take no account of variation according
to position in the blastema; only very rough and somewhat dubious estimates of
the cell cycle parameters can be deduced, except for the length of the G2 + ^M
phase, which turns out to be very short. Our experiments provide new information about the pattern of cell division in the regeneration blastema.
Our study also, however, has its limitations. The pulse-labelling index
measures only the proportion of cells in S-phase; to infer the growth rate one
must make some questionable assumptions: the number of pulse-labelled cells
will be directly proportional to the number of new cells produced by mitosis per
unit time if every S-phase that occurs has the same duration, and is followed by
mitosis after a constant interval. As for continuous labelling, we have not made
an exhaustive study varying the cumulative time of incorporation. Rather, we
compare the effect of a single pulse with that of one standard period of continuous incorporation, and so try to assess the proportion of cells engaged in the
division cycle.
Cell division during growth of blastema
35
MATERIALS AND METHODS
Adult newts of the species Triturus cristatus were used throughout. They were
kept in tanks containing tap water heated to 25 °C, and were fed weekly on
minced heart. To obtain the regeneration blastemas, amputations were made
through both forelimbs, at the mid radius/ulna level. The animals were then left
for varying lengths of time to regenerate to the different stages used in the
experiment. At least two animals of each stage were sectioned and counted.
When the blastemas reached the required stages, the newts were injected
intraperitoneally with 50 fid tritiated thymidine dissolved in 0-05 ml of sterile
water. The isotope, purchased from the Radiochemical Centre, Amersham, had
a specific activity of 24-5 Ci/mmol. For the continuous labelling experiments,
animals were injected with the same dose as in the pulse-labelling experiments
every 12 h for 3 days.
The blastema, plus a small piece of stump, was cut off 2 h after the injection
in the pulse labelling experiments, and 12 h after the final injection in the continuous labelling experiments, and was fixed in half-strength Karnovsky
cacodylate-buffered formalin/glutaraldehyde mixture overnight (see Karnovsky,
1965). The tissue was dehydrated through a graded series of alcohols and embedded in Araldite. Pairs of slides were made of 1 /im thick sections at intervals
of 30 /.im passing through the block, in a longitudinal plane. One of the pair was
used for autoradiography, whilst the other was stained with 1 % toluidine blue
(in 1 % borax) and used as a reference slide, to show the level of the corresponding autoradiograph in the regenerating blastema. The sections for autoradiography were stained by the Feulgen technique.
The Feulgen-stained slides were then used for the autoradiography. This was
carried out employing the dipping technique modified from Kopriwa, Messier
& Leblond (1960) using llford K 5 Emulsion. After the slides had been dipped
and dried, they were stored in light-tight boxes at 4 °C for an exposure time of
one week. They were then developed in D 19 developer for 5 min at 20 °C,
fixed, dried and mounted in Euparal.
Several sections of each stage were photographed at a magnification of about
x 100, on a Leitz photo-microscope. For each section a photomontage was
made, and divided into squares; each square represented 8400 /an 2 except on the
small-cone sections, where each square represented 2100/*m2. Labelled and
unlabelled nuclei were counted in each square in the mesoderm. The ectoderm
appears as a thin strip in section; rather than divide it into squares, we divided it
into segments 100 nuclei long for counting. The mean grain count over labelled
nuclei sectioned through their midriff was about 30. A count of three or more
grains was taken as the criterion of labelling. Proportionately small grain counts
occur over nuclei sectioned peripherally, and so appearing as small fragments.
We excluded from our counts, both of labelled and unlabelled nuclei, those
fragments whose area was less than about one tenth of the full size, since the
36
A. R. SMITH AND A. M. CRAWLEY
mean number of grains to be expected over these fragments, if labelled, would
be less than three. Since our sections were only 1 /im thick, we judged it unnecessary to correct our counts for overlap of cells or self-absorption.
The results are displayed in tables, graphs and contour maps. The contour
maps show data smoothed by a simple computer programme; if the numbers
are plotted raw, the random fluctuations from one small square to the next
obscure the general trend. The basic smoothing procedure consisted in taking
at each vertex of the square grid a mean of the numbers in the four surrounding
squares. This procedure was iterated until it gave a contour map which looked
free from random irregularities - admittedly a subjective judgement, but the
pattern was not very sensitive to the number of iterations beyond the first few.
Figs. 3, 4(a) and 5 (a) show results of the sixth iteration. The numbers appearing
at the sixth iteration are in effect averages of the raw figures for overlapping
groups of 7 x 7 small squares; but the peripheral squares of the group enter the
average with a small statistical weight, and, as one can easily calculate, over
60 % of the weight goes to the central 3 x 3 squares of the group.
RESULTS
Staging
The regeneration blastema of the Italian or crested newt {Triturus cristatus)
passes through stages that appear to differ slightly from those of the American
or spotted newt {Triturus viridescens), which is more commonly used, and so we
introduced our own staging system (Smith et al. 1974). Here we present histological data relevant to our stages.
Small cone (Fig. 1 a). This stage is reached about 10-14 days after amputation.
The wound epithelium has covered the amputation surface and the blastema
cells have started to pile up underneath this, making a small mound whose
height is less than its basal diameter. At this stage, the mesenchyme is undifferentiated and composed of a homogeneous mass of cells.
Large cone. This stage is reached about 2 days later, and is achieved by an
elongation of the cone, so that the height is approximately the same as the basal
diameter. Internally there are some signs of proximal differentiation but this is
not extensive. Proximally, the mesenchyme cells close to the central axis of the
limb start to flatten slightly, at right angles to that axis.
Flat cone (Fig. \b). This stage is normally reached by days 14-18, and is the
result of a general dorso-ventral flattening of the large-cone blastema. Differentiation is now clearly seen internally with the appearance of precartilage in
the form of closely stacked flattened cells running down the middle of the
blastema from the bone of the stump to the distal tip. Cartilage matrix is just
starting to be secreted, but does not stain metachromatically.
Spatulate. This stage is reached about one day after the flat cone and is a
Cell division during growth of blastema
500/im
500/zm
500 nm
Fig. 1. (a) Small-cone blastema, showing homogeneous mass of mesenchyme cells
and thickened apical ectodermal cap. (b) Flat-cone blastema, showing the central
mesenchyme starting to condense to form pre-cartilage. (c) Two-digit blastema,
showing the cential rod of cartilage.
37
38
A. R. SMITH AND A. M. CRAWLEY
Fig. 2. A typical autoradiograph from the ectoderm/mesenchyme border. Background counts are very low.
result of elongation (cf. palette stage of Triturus viridescens). It is during this
stage that the cartilage matrix first stains metachromatically with toluidine blue.
Two digits (Fig. 1 c). This stage is reached by about 3 weeks. An indentation
appears externally, which separates the first two digits. The blastema is still
elongating and widening. Internally the carpals are forming by the splitting up
of the diffuse cartilage mass which lies beyond the regenerated distal ends of the
radius and ulna. This has been described by Stocum & Dearlove (1971).
Muscle ^differentiation was not observed during the course of this study but
elongated cells are seen to line up lateral to the two rods of cartilage.
Pulse labelling
Fig. 2 shows a typical autoradiograph. The labelling index at a given stage
was found to vary considerably depending on which section was chosen. We
have used central sections for our study of blastemal growth. The position of the
stump boundary was recognizable from the position of the dermal glands.
As can be seen from Table 1, the pulse-labelling index of the mesenchyme in
the whole blastema falls with the passage of time from a maximum of over 30 %
Distal half
37-0 ±2-1
39-4 ±1-2
28-9± 11
24-7 ±0-7
19 5 ±2-5
Whole blastema
31-9+ 1-3
27-2 ±0-8
23-6±l-O
21-1 ±0-8
19-5 ±2-3
Stages
Small cone
Large cone
Flat cone
Two digit
Non-cartilage
Cartilage
16-8± 1•1
19-7 + 2•7
26-6 ±2 •5
20-0 ± 0• 8
18-8+1 •0
Pioximal half
27-7± 11
10-3 ±1-5
42-1 ±1-8
33-7 ±1-3
Area 1
21-5 ±0-8
24-7 ±0-5
360 ±0-8
22-5 ±2-5
Area 2
Labelling index
Table 1. Pulse labelling
18-7± 1-8
16-7 ± 5 0
22-2 ±0-9
20-6 ±1-8
Area 3
14-9 ±1-8
17-4+ 1-9
17-9± 1-3
15-4±0-8
Area 4
15-7±O-5
15-7 ±0-5
25-4 ±2-9
21-5 ±2-0
18O±O-8
Ectoderm
vo
Cell divisicm during growth of b
40
A. R. SMITH AND A. M. CRAWLEY
46 \ 44
42
Fig. 3. Computer print-out (third smoothing) of the spatial pattern of the percentage
pulse labelling index of a small-cone blastema. There is no significant proximodistal gradient.
for the small cones down to just over 20 % for the two-digit blastemas. This
drop occurs over a period of about 8 days. The overall density of the mesenchyme
(cells/unit area) does not seem to alter appreciably during the time-course of
this experiment, remaining fairly stable at about 28 cells/10000 /on2.
There are also regional differences in the pattern of labelling: in general the
pulse-labelling index falls off with distance from the tip. In one small cone
blastema the labelling was relatively uniform, whereas in another there was a
clear proximo-distal gradient. All later stages showed a definite proximo-distal
gradient of labelling, with a relatively high index at the distal tips (see Figs. 3-5),
the distal values being about twice the proximal values. However, the absolute
value of the index decreases with time even at the tip.
At the two-digit stage when cartilage was evident (Fig. 1 c) a comparison was
made between cartilaginous and non-cartilaginous regions, and no clear difference in pulse-labelling index was found except near the tip where the precartilage mesenchyme had a much lower index. Spatial variations of cell density
are not observed until the appearance of the cartilage in the central part of the
blastema. Later, the sides of the blastema, particularly in the proximal regions,
become more densely populated as the future muscle begins to form.
From small-cone to the two-digit stage the overall labelling index of the
ectoderm of the stump is raised near the plane of amputation; in the ectoderm
of the blastema, the index is low proximally and high distally, except for a
region of low labelling at the extreme tip. Thus there appear to be two growth
bands of higher labelling index - one around the stump, and the second in the
distal half of the blastema, but proximal to the tip (see Fig. 6).
For a comparison, tritiated thymidine was also injected into normal adult
Stump
Stump
Distal tip
Fig. 4. (A) Computer print out (third smoothing) of the spatial pattern of the percentage pulse-labelling index of a large-cone blastema. A clear proximo-distal
gradient can be seen. (B) the proximo-distal change in pulse-labelling index (O), and
cell density ( • ) of a large-cone blastema. ('Unit area' = 8400 /«n2.)
42
A. R. SMITH AND A. M. CRAWLEY
A
\
33
34
33
35
20
15
Stump
Distal tip
Fig. 5. (A) Computer print-out (third smoothing) of the spatial pattern of the percentage pulse-labelling index of a two-digit blastema. The gradient is not so steep
here, and the pattern is complicated by the presence of mature cartilage. (B) The
proximo-distal change in pulse-labelling index (O) and cell density (#) of a twodigit blastema. Cartilage extended about half-way into the blastema, i.e. as far as
the main dip in the labelling curve. ('Unit area' = 8400/*m2.)
43
Cell division during growth of blastema
117
24-6
15-8
18-4
18-5
Fig. 6. The percentage pulse-labelling index of the ectoderm of (A) a large-cone
blastema and (B) a two-digit blastema. Two bands of high labelling index are seen,
one close to the plane of amputation and the other, more marked, in the distal half
of the blastema, but proximal to the tip.
newts and sections of intact mature limbs were cut. The labelling of these was
very low compared to the regenerates. The mesenchyme had less than 5 %
labelled cells (which were clustered together in small regions). The ectoderm was
evenly labelled at a level of 8-0 + 0-5 %. This should be contrasted with the stump
tissue after amputation, where there was a considerably higher labelling index.
Continuous labelling
After continuous labelling for 3 days (Table 2), only about 50 % of the cells
in the small cone are labelled. At later stages a much higher percentage, usually
over 80 %, of the cells are labelled at the tip, and there is again a proximo-distal
gradient, both in non-cartilaginous and in cartilaginous regions. There is,
however, a peculiarity in the behaviour of the cartilage: its continuous labelling
index drops temporarily in the flat-cone/two-digit stage to about half the value
observed in the corresponding regions earlier and later. The ectoderm was
found to be constantly labelled for all stages at just under 60 %.
DISCUSSION
For a cell population which is not growing steadily, and may not be homogeneous, it is hard to interpret labelling indices rigorously. A host of unknown
variables complicate the analysis; cells may be leaving or entering the population by death or migration; some cells may be quiescent and never divide;
cells that do divide may differ as to the time they spend in each phase of the cell
cycle, and they may differ either randomly, or according to some cryptic
hereditary determination; the average lengths of the phases of the cell cycle may
change as regeneration proceeds; the cells may divide in partial synchrony,
70-5 ±0-4
710±0
76-0 ±3-8
35-3 ±100
82-3 ±4-7
850±l-4
71-0 + 0
56-5 ±5-6
260 ± 6 0
63-3 ±4-4
520 ±5-4
580 ±3-3
45-7 ±2-9
49-3 ±5-7
19-8 ±4-2
500 ±4-2
53-3 ±2-4
51-3±21
Small cone to
large cone
Large cone to
flat cone
Flat cone to
two digit
Non-cartilage
Cartilage
Post two-digit
Non-cartilage
Cartilage
Proximal half
Distal half
Whole blastema
Stages
86-3 ±3-6
980 ±0
83-O±3-8
590 ±0
83-O±O-7
—
Area 1
80-0 ±5-2
82-0 ±0-7
75-3 ±4-5
31-5±101
660 ±0-7
—
Area 2
Labelling index
Table 2. Continuous labelling
73-7±6-2
500 ± 2 1
45-3 ±4-6
42-7 ±4-4
380 ±6-4
14-8 ±2-7
710±0
77-0 ±0-7
54-5 ±8-2
26-3 ±7-2
—
Area 4
—
Area 3
580 ±0
580 ±0
54-5 ±0-4
54-5 ±0-4
57-5 ±0-4
580 ±0
Ectoderm
o
X
H
hH
on
r
^. M. CR AWLEY
Cell division during growth of blastema
45
especially in the first cycle after amputation. All these possibilities are plausible.
Each of them affects differently the relationship between the labelling index, the
time of labelling, and the parameters of cell proliferation. They are all entangled
with one another in that relationship. One might write down a formula for the
labelling index based on the most general assumptions, and perhaps by dint of
arduous and complicated mathematics invert it, so as to express all the unknowns
as functions of labelling indices. Since the unknowns are not simply a finite set
of numbers, but include whole probability distributions and functions of time
of unknown form, one could not hope to determine them fully except by a full
and accurate specification of the labelling index at every stage and, presumably,
for every possible period of continuous labelling. The index will of course vary
from place to place over the regenerate, and randomly from newt to newt. All
in all, this presents the experimenter with a daunting task, which we have not
undertaken. Instead, we wish to present our more limited data as a baseline for
future studies of, for example, the effects of X-irradiation.
The results we have obtained are substantially in accord with those of earlier
workers such as Litwiller (1939), Chalkley (1954, 1956) and Hearson (in
Thornton, 1968), all of whom found a distal peak of mitotic index in the
mesenchyme. Our results do not accord with those of Hay (1966), who also used
an autoradiographic approach; she maintained that the growth rate was higher
proximally than distally at the 'cone' stage. There is now, however, almost
overwhelming evidence that the structures are laid down in a proximo-distal
sequence, as in the chick limb (Saunders, 1948; Summerbell et al. 1973): the
findings of Stocum (1968 a, b) and Stocum & Dearlove (1971) in particular are
compelling. The original suggestion of Faber (1965) that distal regions are
specified first is no longer tenable (see Faber 1971). There is a superficial conflict
with some of Chalkley's graphs (see Chalkley, 1954, 1956), which show the
mitotic index of the mesenchyme falling abruptly to zero at the extreme tip.
Closer inspection of his figures reveals that this is merely an artifact, because
at the place which Chalkley takes as the tip, the area of the cross-section scanned
for mitoses is very small, if not actually zero. So few cells lie in that area that the
mean total number of mitoses which Chalkley might have expected to count
would be much less than one, even if the rate of proliferation there were as high
as anywhere else in the limb. In other words, the cell population counted at the
tip is too small to yield any useful estimate of the mitotic index. As mentioned
in the Introduction, this is a characteristic defect of mitotic index studies. The
proper policy, in the case of Chalkley's data, seems to be to assume for the
extreme tip the same mitotic index as for the closest tissue for which a reliable
estimate is available - i.e. the same as for the section just proximal to the tip.
This course has been taken in calculating fate maps and population growth
rates from Chalkley's otherwise excellent data (Julian Lewis, in preparation).
With this correction, Chalkley's figures, like ours, show the tip as a region of
rapid cell proliferation.
4
EMB
37
46
A. R. SMITH AND A. M. CRAWLEY
The progress zone model assumes a proximo-distal sequence for the specification of the elements during regeneration and requires a distal growth zone.
Moreover, if change in positional value in that zone is linked to cell division as
our model suggests, one would expect all the cells there to be dividing. Our
results are on the whole consistent with such a model. From large-cone onwards
the cells in a region 300 /«n from the tip, which possibly corresponds to the
progress zone, have the highest labelling index, and more than about 80 % of
them are dividing.
In the small cone only about 50 % of the cells appear to be dividing. Perhaps
the progress zone is not yet fully established, so that some cells which will
eventually be stimulated to divide are still quiescent; alternatively, it could be
that the population of the progress zone is initially contaminated with cells which
can never divide and which will therefore make only a negligible contribution to
the regenerate at later stages. The space occupied by the progress zone may
decrease in the later stages; for example, in the two-digit stage it would lie in the
mesenchyme distal to the two rods of cartilage. Possibly after the two-digit
stage, the progress zone would persist only on one side in the area where the
third and fourth digits form. The progress zone must remain until the last
digital element has been laid down.
The overall pulse-labelling index falls with time, dropping faster in the proximal regions as they differentiate, so that a gradient of labelling index is set up.
This is similar to that found by Hornbruch & Wolpert (1971), who showed a
gradient of mitotic index in the chick wing-bud. However, the blastema was
different from the wing-bud in that we could find no significant correlation
between density and labelling index in it, as checked using scatter diagrams (see
Figs. 4b, 5 b).
The anomalously low continuous-labelling index of the cartilage at the flat
cone/two-digit stage suggests a parallel with the embryonic chick limb, in which
the precartilage cells likewise pause in their programme of proliferation (Julian
Lewis, unpublished). This behaviour may be expected to yield a rather complicated pattern of spatial and temporal changes of labelling index in the developing cartilage. Thus it is perhaps not surprising that the pulse-labelling index for
the cartilage at the two-digit stage does not show a regular proximo-distal
gradient. Our data, however, are not adequate to resolve the details of these
changes of proliferation rate in the cartilage.
The ectoderm-labelling index also falls with the passage of time but shows
regional differences in that two bands of high labelling index are formed - one
around the stump and the second in the distal half of the blastema, but proximal
to the tip (see Fig. 6). It is interesting to note that Hay & Fischman (1960, 1961),
in their study of blastema formation, found the apical cap to have a low labelling
index. However, as in most other work on labelling as a measure of cell division,
no quantitative results were reported.
In a previous paper, we showed that the measured growth curves of regenera
Cell division during growth of blastema
47
tion blastemas could be quite accurately fitted by predictions based on the
progress zone theory of limb development. The predictions involved several
disposable parameters; we chose their values so as to give a good fit to the
experimental growth curves. The present autoradiographic data yield rough but
direct estimates of some of those parameters, and provide a check on the
plausibility of some of the assumptions we made before.
Our previous calculation dealt with the rate of elongation of the parts of the
blastema along the proximo-distal axis. Autoradiography gives information
about the rate of growth of the cell population. This will be proportional to the
rate of elongation if (1) the number of cells per unit volume, or 'cell density',
remains constant and (2) outgrowth occurs strictly by elongation, and is not
accompanied by changes of cross-sectional area. If part of the increase of the
cell population is taken up by an increase of cell density or of cross-sectional
area, the rate of elongation will be reduced. In practice, the cell density seems to
be roughly constant, and the cross-sectional area grows only slightly during the
course of this study.
For the stages from large cone to two digit, between 70 % and 90 % of the cells
at the tip are labelled at the end of 3 days of continuous labelling. This is compatible with the assumption that practically all the cells at the tip are engaged in
the division cycle, with a cycle time, and hence a population doubling time, of
slightly over 3 days. In our previous calculation, by comparison, we assumed
that the intrinsic growth rate in the progress zone at the tip was g = 0-16 days"1,
i.e. the tissue there was elongating at a rate of 16 % per day. This corresponds
to a length-doubling time of In 2/0-16 days, i.e. 4-3 days.
We assumed, furthermore, that this growth rate remained constant at the tip
from the small-cone to the two-digit stage. For confirmation, one can turn now
to the autoradiographic /w/se-labelling index: with provisos, it should be
approximately proportional to the rate of growth of the cell population. The
provisos are (1) that the lengths of the S-phase and of the G2 phase stay constant
and (2) that changes in the rate of population growth arise solely from changes
in the length of Gl5 and/or from withdrawals from Gx into a non-proliferating
Go state. The pulse-labelling index near the tip is indeed nearly constant: 37 %
at small cone; 42 % at large cone; 34 °/0 at flat cone; 28 °/0 at two digit.
We would like to thank Professor Lewis Wolpert and Dr Julian Lewis for their help and
encouragement. This work was supported by the Medical Research Council.
REFERENCES
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FABER, J. (1965). Autonomous morphogenetic activities of the amphibian regeneration
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Trampusch), pp. 404-419. Amsterdam: North-Holland.
CHALKLEY,
4-2
48
A. R. SMITH AND A. M. CRAWLEY
J. (1971). Vertebrate limb ontogeny and limb regeneration: morphogenetic parallels.
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GRILLO, R. S. (1971). Changes in mitotic activity during limb regeneration in Triturus.
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HAY, E. D. & FISCHMAN, D. A. (1961). Origin of the blastema in the regenerating newt
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HORNBRUCH, A. & WOLPERT, L. (1971). Cell division in the early growth and morphogenesis
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{Received 25 April 1975; revised 28 July 1976)