/. Embryol exp. Morph. Vol. 63, pp. 145-159, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Positional signalling along the anteroposterior
axis of the chick wing. The effect of multiple
polarizing region grafts
By L. WOLPERT AND AMATA HORNBRUCH1
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School, London
SUMMARY
We have proposed that positional information along the anteroposterior axis is specified
by a signal from the polarizing region and that position may be specified by the concentration
of a diffusible morphogen. While this model can account for a variety of results it is t>ow
clear that a model based on intercalation by growth of positional values can do the same.
The distinction between the two models lies in whether a grafted polarizing region can alter
existing positional values and in the distance over which it exerts its influence. The two
models make different predictions as to the effect of grafting two polarizing regions. The
intercalation model predicts that this effect will be the sum of two single grafts, whereas the
morphogen model predicts different results depending on how close together the two polarizing regions are placed. The pattern of digits following grafts of two polarizing regions show
that it is sensitive to the distance between the grafts and consistent with a model based on
long-range interaction, such as a diffusible morphogen.
INTRODUCTION
The pattern of cellular differentiation in the chick limb has been considered
in terms of positional information (Wolpert, Lewis & Summerbell, 1975). For
the anteroposterior axis we suggested that position is specified with respect to a
region at the posterior margin of the limb, the zone of polarizing activity, originally discovered by Saunders & Gasseling (1968) (reviewed, Tickle, 1980).
Our model proposed that a signal from this region set up a gradient of positional
information along the anteroposterior axis. More specifically, we suggested a
mechanism based upon diffusion of a morphogen from the polarizing region
(Tickle, Summerbell & Wolpert, 1975). It was assumed that the morphogen
was broken down at a rate proportional to its concentration, and thus an
exponential concentration gradient with its highpoint at the posterior margin
was set up. If the signal from the polarizing region was a diffusible morphogen,
it could be expected to diffuse across a substantial portion of the limb bud.
1
Author's address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London W1P 6DB, U.K.
J45
146
L. WOLPERT AND A. HORNBRUCH
Following a graft of an additional polarizing region, the new distribution of the
morphogen was assumed to assign to the cells new positional values. Using the
digits as markers for different positions along the anteroposterior axis, it was
shown that this model could reasonably account for the pattern obtained when
an additional polarizing region was placed at different positions along the
anteroposterior axis. An important proviso was that the limb bud widened
prior to digit specification (Tickle et ah 1975; Summerbell & Tickle, 1977).
Recently, Summerbell (1979) has provided further evidence for such a gradient
in the early limb bud. He inserted barriers at different positions along the anteroposterior axis and found that the defects obtained were consistent with position
being specified by a diffusible morphogen. Further evidence has also been provided by MacCabe & Parker (1976) and Smith, Tickle & Wolpert (1978).
However, following discussion with Drs Susan Bryant and Laurie Iten, it has
become apparent that many of the results obtained by grafting on additional
polarizing regions to the developing wing bud could be accounted for on a rather
different model, intercalary regeneration, similar to that observed in insect
imaginal discs and cockroach legs (French, Bryant & Bryant, 1976). The essential
mechanism is that when tissues are grafted such that non-contiguous positional values are opposed, growth occurs at the junction and new positional
values are generated until the discordance is no longer present (see Iten &
Murphy, 1980).
The difference between the two models can be illustrated by how they account
for the pattern of digits when a polarizing region is grafted at the anterior
margin, or if it is placed near the tip of the limb. When it is placed at the anterior
margin opposite somite 16, the pattern of digits is 4 3 2 2 3 4, and when at the
centre of the limb the pattern is typically 2 3 4 4 or 2 3 4 4 3 4 depending on its
precise position.
For the intercalary model in Fig. 1, it is assumed that there is an even distribution of positional values along the anteroposterior axis of the limb bud. The
three digits, 2, 3 and 4 are considered to form opposite somites 18 and 19 and
they are thus assigned positional values 5, 6/7 and 8 respectively, which is more
or less in line with Summerbell (1979). When a polarizing region is grafted to
different positions along the anteroposterior axis such that non-contiguous
positional values are adjacent to one another, intercalary growth occurs so as
to generate the missing values. Thus, when the polarizing region, which has a
positional value of 10 is placed at the anterior margin, there is intercalation
between the graft positional value 10 and the adjacent host value of 3. Thus, a
complete new set of digits is formed (Fig. \b). When the graft is opposite
somite 18, the predicted result is 2 3 4 4 4 (Fig. 1 c) though experiments show that
one of the 4's is usually absent. The model can thus reasonably account for
grafts of the polarizing region to different positions along the anteroposterior
axis.
The model for a diffusible morphogen providing the positional signal assumes
Positional signalling along anteroposterior axis of chick wing
12345678910
2 3 4
147
Positional values
Digits
Polarising region
( H 3 4 5 6 7 8 9 10 Positional values
I
[T0l98 7 6 5 4 3 4 S f i 7 K Q i n
Positional values after
intercalation
4 3 2
2 3 4
Digits
1 2 3 4 5[Tg 7 8 9 10
Positional values
1 2 3 4 5 6 7 8 9 I T 0 1 9 8 7 8 9 10 after intercalation
2 3 4
4
4
Digits
[10] 3 4 SffOl 7 8 9 10
5
Positional values
ITD19876 5 4 3 4 5 6 7 8 9fTo]9 8 7 8 9 10 after intercalaction
4 3 2
2 3 4
4
4
Digits
Fig. 1. Diagrams to illustrate the pattern of digits to be expected in terms of a model
based on intercalation. It is assumed that, as in (a), there is a set of positional values
along the anteroposterior axis and that the digits develop at particular positional
values. For example, digit 2 forms at positional value 5. When grafts are made of
polarizing region, whose positional value is 10, to different positions along the
anteroposterior axis, intercalation occurs, and the pattern of digits will be determined
by the resulting positional values. When the polarizing region is grafted opposite
somite 16 as in (b) intercalation occurs to give 4 3 2 2 3 4. When grafted opposite
somite 18 (c) the pattern after intercalation is 2 3 4 4 4. In (d) polarizing regions
are grafted opposite somites 16 and 18, and the resulting pattern of digits is 4 3 223444.
Concentration of morphogen
Concentration of morphogen
Concentration of morphogen
o
o
>
r
3
o
4
oo
Positional signalling along anteroposterior axis of chick wing
149
Anterior
10 0 I
i
i
i
~
i
I
I
p p
p p p p o o Q
o o o o o o ^ o
L
— — -70 0 0
Distance
Anterior
Posterior
100T (e)
4 o
Distance
Fig. 2. (o) The concentration of a postulated morphogen, produced by the polarizing
region, along the anteroposterior axis. Digits are specified at different concentrations of the morphogen. In (b) a polarizing region has been grafted opposite somite
16 and the resulting pattern of digits is 4 3 2 2 3 4. In (c) the polarizing region has
been grafted opposite somite 18 and the pattern of digits is 23 4 4 4 . Note the
similarity of pattern of digits in (b) and (c) with those predicted by the intercalation
model in Fig. 1 (b) and(c).In(d)polarizingregions have been grafted opposite somites
16 and 18 and the pattern of digits is 4 3 3 44 4 and is different from that predicted by intercalation in Fig. 1 (d). In (e) polarizing regions are grafted opposite
somites 16 and 17 and the pattern of digits is 4 4 3 3 4.
The expression for the concentration of the morphogen for two sources distance
d apart is
CSOurce sinh P(d— x) + sinh Px
sinh Pd
'
C source has been taken as 100, and P which reflects morphogen breakdown and
diffusion constant, as 70.
150
L. WOLPERT AND A. HORNBRUCH
that the polarizing region is a source which keeps the concentration of the
morphogen at a constant value of 100 (Fig. 2). The substance is assumed to be
broken down at a rate proportional to its concentration, thus giving an exponential gradient. It is important to note that once the position of the digits is
fixed, only one parameter can be varied. This is the relation between the diffusion
constant and the rate of breakdown of the morphogen and it determines the
steepness of the morphogen gradient. We have chosen a gradient such that the
thresholds for digits 2, 3 and 4, are morphogen concentrations 4, 15 and 45
respectively. Following grafting of a polarizing region, the limb widens 50 %
at 36 h after the graft (Tickle et al. 1975; Smith & Wolpert, 1980). This is
the time at which the digits begin to be laid down (Summerbell, 1974). Thus,
in calculating the new distribution of the morphogen following a polarizing
region graft, we have assumed that the distance between the polarizing regions
has increased by 50 %. As can be seen, this model gives similar predictions for
the standard grafts, but it should be regarded as no more than semi-quantitative
since numerous assumptions such as diffusion in a single dimension and point
sources, are made. A more biochemically realistic gradient without point
sources could be generated using the mechanism proposed by Meinhardt &
Gierer (1974).
The essential difference between the two models is that between morphallaxis
and epimorphosis (Wolpert, 1971). In morphallaxis, positional values are
changed and interaction and signalling may occur over most of the field,
whereas with epimorphic regulation, all existing positional values are retained
and new ones generated by growth (Wolpert, 1971; Cooke, 1979). In terms of
the particular case under consideration, the distinction lies in two important
processes: (1) The change in positional values adjacent to the polarizing region.
If there is signalling as in morphallaxis, then one would expect the polarizing
region to alter the positional values of the cells adjacent to it. With an epimorphic process, all the existing positional values should be retained and the
new ones generated by growth. No positional values should be lost (see Fig. 1).
(2) The distance over which the polarizing region exerts its influence. In the
case of morphallaxis it would signal over about 400 /^m altering the positional
value of adjacent cells (Fig. 2). In epimorphosis, in principle at least, the signal
from the polarizing region need not extend more than one cell diameter.
In drawing the distinction between morphallaxis and epimorphosis, it is the
processes involved that are important and not any absolute distinction. As
Cooke (1979) has pointed out, both may be involved in a particular system.
Moreover, even in epimorphosis, the distance over which the interactions occur
may be quite extensive. Thus, the important questions are whether the polarizing region alters existing positional values and over what distance it exerts its
influence.
We have approached these problems by grafting two additional polarizing
regions to different positions in the limb bud. As pointed out above, a single
Positional signalling along anteroposterior axis of chick wing
151
polarizing region grafted to the anterior margin - opposite somite 16-gives
4 3 2 2 3 4, whereas if it is grafted opposite somite 18, it typically gives 2 3 4 4 .
Both results are explicable in terms of the two models. What will happen if the
two grafts are made in the same limb? What structures will develop between the
two grafted polarizing regions? According to an intercalation model the results
sould be additive, giving 4 3 2 2 3 4 4 since no positional values would be lost
(Fig. Id). Most important, when one polarizing region is opposite somite 16
then wherever the other polarizing region is placed, digits 4 3 2 2 3 4 should
always form between the two grafts. However, on a signalling model the
prediction would be that 4 3 3 4 form between the grafts since the two polarizing
regions would be so close to each other that the concentration of the morphogen
would be too high to allow a digit 2 to form between them (Fig. Id).
We have thus explored the effect of grafting one polarizing region opposite
somite 16 and a second polarizing region at different positions along the
anteroposterior axis.
The model we have analysed in Fig. 1 is based on linear intercalation, and is
thus similar to that proposed by Bohn (1970) for the proximodistal axis of the
cockroach leg. Since Iten & Murphy (1980) have suggested that the polar coordinate model may be more appropriate, it is necessary for us to explain why
we have not used it. Our linear intercalation model largely accounts for the
standard results, whereas, as yet, no polar coordinate model has been put forward to account for the results of the polarizing region grafts. The diagnostic
features of the polar coordinate model in other systems are regeneration,
duplication, and incomplete distal transformation when a complete circle of
circumferential values is not present (French et al. 1976.) None of these have
been demonstrated in the chick limb bud. For example, removal of posterior
tissue often leads to the loss of digit 4 (Tickle et al. 1975: Fallon & Crosby,
1975). In a polar coordinate system regeneration, or duplication, should result.
As has been pointed out by Fallon & Crosby (1975) the supernumerary structures that form when the tip is rotated 180 ° are dependent on the polarizing
region. By contrast dorsoventral inversion of the tip does not lead to the formation of supernumerary structures (Saunders, Gasseling & Gfeller, 1958) as
required by the polar coordinate model.
The results of Iten & Murphy (1980) do not demand a polar coordinate
model for their interpretation and, in fact, on the whole, are consistent with the
long-range signal model. Their approach has been to graft anterior margin
tissue as a wedge to the polarizing region.They found that this results in supernumerary limb structures being formed. They recognise that this can be understood in terms of a signal to the graft from the host polarizing region, but
point out that anterior margin tissue gives more structures than tissue taken
from a slightly more posterior position, and this is not predicted with such a
model. The difference is quite small and is essentially that tissue from opposite
somite 16/17 results in an additional 4 3 whereas tissues from opposite 17/18
152
L. WOLPERT AND A. HORNBRUCH
gives only 3. We have no explanation for this difference but do not regard it as
evidence for a polar coordinate model. In fact, no explanation is offered by
Iten & Murphy in terms of their model.
Both a linear intercalation model and a polar coordinate model would be
expected to give similar results when anterior tissue is confronted with posterior
margin, whether the anterior margin is grafted to a posterior position or whether
posterior margin is grafted anteriorly. This is not the case since the former
invariably results in an extra digit 2, whereas the latter rarely does according to
Iten & Murphy (1980). This is to be expected in terms of our model since the
graft will be too close to the polarizing region to allow a digit 2 to form.
Finally it is very difficult to understand attenuation in terms of a polar
coordinate model. Attentuation of the signal from the polarizing region has
been demonstrated in several ways. Smith, Tickle & Wolpert (1978) showed that
when the polarizing region was subjected to increasing doses of y-irradiation,
the 'highest' new digit specified changed from 4 to 3 to 2. Tickle (1981) has
shown the same phenomenon both by diluting the polarizing region grafted
with non-polarizing region cells, and by grafting small numbers of cells in a
monolayer.
MATERIALS AND METHODS
Fertilized White Leghorn eggs from a local breeder were incubated at 38 ± 1
°C and windowed on the fourth day of incubation. The embryos were staged
according to Hamburger & Hamilton (1951). Embryos at stages 19 and 20
were chosen as hosts and polarizing regions were taken from donors at stage
21. The polarizing region was excised from opposite somite 20 and measured
not more than 150 by 150 /im. The site for the graft was prepared by removing
a cube of tissue the same size as the graft from along the anterior margin of the
host limb bud. The graft was kept in place with a platinum wire pin. As controls,
anterior margin tissue from opposite somite 16 was used for grafting in the same
manner. For some grafts of polarizing region, no material was removed, but
following Iten & Murphy (1980) a slit was made in the host and a wedgeshaped piece of tissue from the polarizing region inserted.
Six days after the operation the limbs were fixed in 5 % trichloracetic acid,
stained with Alcian green (Summerbell & Wolpert, 1973), cleared in methyl
salicylate and examined.
RESULTS
The results of grafting two polarizing regions are shown in Table 1, and
Table 2 has been constructed from Table 1 by determining the pattern of the
digits formed between the grafted polarizing regions. The results in Table 2
show the distribution of the 'lowest' digits found, digit 2 being 'lower' than
digit 3. In constructing Table 2 from Table 1 it was necessary to infer where the
polarizing regions had been placed. In some cases this is quite easy where
Positional signalling along anteroposterior
axis of chick wing
153
Table 1. Pattern of digits following grafts of two polarizing regions
Position of
polarizing
regions
with respect
to somites
Total
16 and 19
13
16 and 18/19
11
16 and 18
Number of
cases
9
16 and 17
8
Posterior
4 3 2 23
4 3 23
3 2 23
2 23
2 3
4 3 2 3
4 3 23
4 3 2 23
3 223
2 23
4 3
3 223
3 23
4 3 23
4 3 3 443
4 33
4 33
4 3
4 34 4 3 3
5
1
1
1
5
2
3
2
1
1
2
1
1
2
1
1
2
1
3
2
3
1
9
16 and 17/18
Anterior
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
344
34
432 34
4334
344334
4334
5
3
Table 2. Digits that develop between two grafted polarizing regions
Position of polarizing regions with
respect to somites
16 and 19
16 and 18/19
16 and 18
16 and 17/18
16 and 17
Wedges
16/17 and 17/18
Digits formed between grafts
r
...22...
?
9
4
1
3
3
3
3
—
—
..33...
3
0
1
4
4
—
1
1
2
5
3
—
4
4
154
L. WOLPERT AND A. HORNBRUCH
•A
^jt
\
y
5(a)
i
(a)
8(6) ^
Positional signalling along anteroposterior
axis of chick wing
155
Table 3. Pattern of digits following grafts of combinations of polarizing
region and anterior margin
Site of grafts
Polarizing region
opposite somite 16
and anterior margin
opposite somite 18
Anterior margin
opposite somite 16
and polarizing region
opposite somite 19
Total
9
9
Number of
cases
4
2
1
2
5
4
Anterior
Posterior
43223
4 3 2 3
3 2
2 3
23
2 3
4
4
4
4
4
digit 4 provides the boundaries. In other cases it is less obvious. However, we
can make use of Summerbells' (1980) results which are substantially similar
and which are based on observation of the limbs following grafting.
From Tables 1 and 2 it can be seen that as the second polarizing region is
placed in successively more anterior positions, the probability of a digit 2
forming between them falls. When it is placed opposite somite 19, the pattern
of digits is unchanged and is usually 4 3 2 2 3 4, as when only polarizing region
is grafted to the anterior margin opposite somite 16. At least one digit 2 forms
between the polarizing regions in every case, and two digit 2s form in most
cases. However, when the second polarizing region is placed opposite somite
18/19, two digit 2s form in only 40 % of the cases, and a digit 2 only develops
80% of the time. When the second polarizing region is opposite somite 18
two digit 2s only form in 12 % of the cases, and no digit 2 at all forms in 56 %
of the limbs. When the polarizing regions were grafted opposite somites 16 and
17, no digit 2 formed at all (Fig. 5).
Fig. 3. Normal wing at 10 days of incubation. The pattern of digits is 2 3 4.
Fig. 4. Mirror image reduplication following a polarizing region grafted opposite
somite 16. The pattern of digits is 4 3 2 2 3 4.
Fig. 5. Limbs that developed following the grafting of polarizing regions opposite
somites 16 and 17. In (a) the pattern of digits is 4 3 4 4 3 3 4 and in (6) it is 4 3 3 4,
note that there are two ulnae.
Fig. 6. Limb that developed following graft of two polarizing regions as wedges
opposite somites 16/17 and 17/18. The pattern of digits is 4 3 4 3 3 4. Note that the
distal humerus is reduplicated and the bud seems to be split into two.
Fig. 7. Limb that developed following a graft of a polarizing region opposite
somite 18.
Fig. 8. Limbs that developed following a graft of a polarizing region as a wedge
opposite somite 17/18. In (a) the pattern of digits is 2 3 4 3 3 4 and there is a
radius with two ulnae. In (b) the pattern of digits is 2 3 4 4 3 3 4 and there is one
radius and three ulnae. It appears in this case that the bud has been split into two*
156
L. WOLPERT AND A. HORNBRUCH
Table 4. Pattern of digits following single polarizing region grafts
Position of polarizing regions with
respect to somites
Total
16
17
17
8
18
8
Number of
cases
10
5
2
1
1
3
1
3
1
1
3
4
Anterior
Posterior
4 3 22 3
4 3 2 3
2 3
2 3 4 3 3
3 4 3 3
3 4 4 3 3
4 3 3
4 32 3
3 2 3
2 3 44 3
2 344
2 3
4
4
4
4
4
4
4
4
4
4
4
Table 5. Pattern of digits following wedge-shaped grafts without
removal of host tissue
Site of grafts
Total
Anterior margin
opposite somite 17/18
Polarizing region
opposite somite 17/18
8
Polarizing regions
opposite both
Number of cases
Anterior
2 34
3 4
6
2
8
3
8
3
1
1
1
1
somite 16/17 and
1
somite 17/18
1
1
2
1
Posterior
2
2
2
2
2
4
4
3
3
3
3
3
3
3
3
3
443
4 3
44
3 4
3 4
3 4
4 43
443
43
43
4 43
4 3
3
2 34
3 4
3 4
3 4
3 4
3 4
3 4
3 4
In all the above grafts, host material was removed when the polarizing region
was grafted. This might affect the results, and so following a suggestion of
L. Iten, polarizing regions were also grafted as wedges, no host material being
removed. When these were grafted opposite somites 16/17 and 17/18, no digit 2
formed between them (Fig. 6). A further control involved grafting a polarizing
region opposite somite 16 and a piece of anterior margin (from opposite somite
16) to opposite somite 18. As can be seen from Table 3, a digit 2 is formed in all
cases though the number of double digit 2s is reduced by half.
Some grafts of a single polarizing region and anterior margin were also
carried out as controls (Table 4). A polarizing region opposite somite 18
Positional signalling along anteroposterior axis of chick wing
157
usually gave 2 3 4, or 2 3 4 4 (Fig. 7). When it was grafted opposite somite 17,
a central digit 2 formed in 40 % of the cases, and when it did not, an anterior
digit 3 usually developed. When a polarizing region was grafted as a wedge into
a slit opposite somite 17/18, there was only one case of a digit 2 centrally,
whereas an anterior digit 2 formed in every case. (Table 5) (Fig. 8).
DISCUSSION
These results show that when two polarizing regions are grafted to the early
limb bud, such that one is at the anterior margin, then the pattern of digits
formed between them depends on the distance between the two grafts (Table 2).
When the two polarizing regions are far apart, two digit 2s usually form between
them. However when one is opposite somite 16 and the other opposite somite
18, two digit 3s is the typical result. When polarizing regions are opposite
somites 16 and 17, no digit 2 forms at all. This is not due to the removal of
material to make place for the grafted polarizing regions since a similar result
is obtained when polarizing regions are grafted, without removing host material
opposite somites 16/17 and 17/18.
These results strongly indicate that an intercalary model of the type outlined
in the introduction is not valid, since it predicts the presence of two digit 2s
between the two polarizing regions in all cases. By contrast, the model based
on a diffusible morphogen accounts for the results reasonably well considering
how idealized it is. The concentration of the diffusible morphogen and thus the
pattern of digits formed is very sensitive to the distance between polarizing
regions. When the initial distance is about 600 /«n or less the concentration
everywhere is too high for digit 2 to be specified.
It is important to remember that all the diffusion curves have been based on a
50 % increase in the distance between the grafted polarizing regions when the
digits are specified. Following a polarizing region graft, widening starts within
about 10 h and the width has increased by 50% within 36 h (Smith &
Wolpert, 1980). If this increase in width is prevented by a low dose of X-irradiation following a polarizing region graft then the pattern of digits will be altered
because the host and grafted polarizing regions will remain closer together. This
should result in the absence of digit 2 and this is in fact what happens (Smith &
Wolpert, 1980).
The effect of polarizing region on growth has been studied by Cooke &
Summerbell (1980) who found a significant increase in labelling with [3H]thymidine within a few hours after grafting. Summerbell (1981) has specifically
studied the effect on growth along the anteroposterior margin using double
polarizing region grafts.
It is of interest to consider the pattern of digits formed when the polarizing
regions are close together as when they are at somite 16 and 17, or in slits opposite somites 16/17 and 17/18. In several cases, as many as 7 digits were obtained
6
EMB 63
158
L. WOLPERT AND A. HORNBRUCH
and this is the maximum so observed. A typical pattern has 4 3 4 4 3 3 4 which
can be understood in terms of the anterior 4 3 4 lying between the two grafted
polarizing regions (Figs 5 and 6). This is clearly illustrated by first considering a
polarizing region graft in a slit opposite somites 17/18 where a typical pattern
was 2 3 4 4 3 3 4. When an additional polarizing region is placed at 16/17, the
anterior 2 is transformed to a 4. It also means that three digits 4 3 4 can form
from about one somite's width of material. One can exclude significant contribution from the polarizing region (Summerbell, 1981). This is consistent with
the presumptive digit pattern of Summerbell (1979) where the three digits in the
normal limb occupy just over a somite's length of limb-bud mesoderm. When
polarizing regions are grafted opposite somites 16 and 17 only digit 4 should be
expected to form between them (Fig. 2e). However, as pointed out in several
cases, a 4 3 4 developed. This may reflect a delay in the diffusion of the morphogen. It should be noted that in the model presented here it is assumed that
equilibrium has been achieved by the time the digits are specified.
An examination of the curves with respect to the idea of thresholds for digits
shows that in some cases much thicker digits, or fused digits, might be expected.
This is not normally the case and digits, particularly the phalanges, are remarkably discrete. However, sometimes the metacarpal of, for example, digit 3 is
very thick, and distally there are two sets of phalanges, Fig. 8 (a). More proximally, it is quite common to find fusion between ulna and radius. It thus seems
that there may be some additional mechanism for keeping digits discrete.
In one case where a polarizing region was grafted opposite somite 16 and a
piece of anterior margin grafted opposite somite 18 a 3 2 4 pattern resulted. This
is the first time we have observed a 2 adjacent to a 4 in over a thousand grafts.
It can be understood in terms of the anterior margin graft causing damage to
the apical ridge and thus loss of the host digit 3.
In conclusion, the results presented here are consistent with the signal from
the polarizing region being a diffusible morphogen which acts over a distance of
several hundred microns. Further direct evidence for such long-range signalling
has been obtained by Honig (1981). He interposed leg tissue between the
grafted polarizing region and the responding wing-bud tissue and showed that
the polarizing region could affect tissue more than 200 /«n away from it.
This work is supported by the Medical Research Council.
We wish to thank Dr C. Tickle for her comments and advice, D. Wolpert for a computer
programme, and Miss M. Maloney for preparing the paper.
REFERENCES
BOHN, H. (1970). Interkalare regeneration und segmentale gradienten bei ein Extremitaten
von Leucophaea - Larven (Blattaria). 1. Femur und Tibia. Wilhelm Roux Archiv. EntwMech. Org. 165, 303.
COOKE, J. (1979). Cell number in relation to primary pattern formation in the embryo
Xenopus laevis. I. The cell cycle during new pattern formation in response to implanted
organizers. /. Embryol. exp. Morph. 51, 165-182.
Positional signalling along anteroposterior axis of chick wing
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(Received 28 July 1980, revised 15 December 1980)
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