/. Embryol exp. Morph. Vol. 63, pp.

127-144, 1981 127

Printed in Great Britain © Company of Biologists Limited 1981

Pattern formation along the anteroposterior

axis of the chick wing: the increase in width
following a polarizing region graft and the
effect of X-irradiation
By J. C. SMITH 1 AND L. WOLPERT 2
From the Department of Biology as Applied to Medicine, The Middlesex Hospital
Medical School, London

SUMMARY
A study is made of the widening of the chick limb bud that occurs after a graft of an
additional polarizing region. Such buds are about 50% wider than controls, after 36 h. By
contrast, growth along the proximodistal axis is unaffected. This widening is reduced by
treating the host embryo with 10 Gy X-irradiation and the altered pattern of digits is
consistent with a diffusible morphogen model for the specification of positional information
along the anteroposterior axis.

INTRODUCTION
Positional information along the anteroposterior axis of the chick wing bud
appears to be specified by the polarizing region at the posterior margin of the
bud. When tissue from this region is grafted to the anterior border of a bud, a
limb with mirror-image symmetry about its long axis is formed (Saunders &
Gasseling, 1968; Tickle, Summerbell & Wolpert, 1975; Fallon & Crosby, 1977;
Summerbell & Tickle, 1977; Smith, Tickle & Wolpert, 1978). A considerable
amount of evidence suggests that the polarizing region acts by producing a
diffusible morphogen, the concentration of which is highest close to the
polarizing region and lower more distant (see Tickle et al. 1975; MacCabe &
Parker, 1975, 1976; Summerbell & Tickle, 1977; MacCabe, Calandra &
Parker, 1977; Smith et al. 1978; MacCabe, Lyle & Lence, 1979; Summerbell,
1979). The responding cells are assumed to be able to interpret the local con-
centration of morphogen and so behave according to their position. For instance
digit 4 forms closest to the polarizing region where the concentration of mor-
phogen would be highest, then digit 3, then digit 2.
One of the earliest responses to a polarizing region graft is an increase in the
1
Author's Address: Laboratory of Tumour Biology, Group W, The Sidney Farber
Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
2
Author's Address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London, WIP 6DB, UK.
5-4
128 J. C. SMITH AND L. WOLPERT
anteroposterior width of the host limb (Tickle et al. 1975; Summerbell &
Tickle, 1977; Fallon & Crosby, 1977). Tickle et al. (1975) and Summerbell &
Tickle (1977) have suggested that this increase in width is of some importance
in the specification of positional information for without it the concentration
of morphogen in the middle of a host wing would not fall low enough to specify
digit 2. In the first part of this paper a study is made of the increase in width
of wing buds to which an additional polarizing region has been grafted. In
the second part it is found that the widening is greatly inhibited by a low dose
of X-irradiation and the pattern of digits that results is consistent with the
diffusible morphogen model since digit 2 is absent.
It should be noted that Iten & Murphy (1980) have suggested that the pattern
of digits following polarizing region grafts may be due to intercalation involving
epimorphosis. This is discussed by Wolpert & Hornbruch (1981) where experi-
ments are presented which suggest that a linear intercalation model is not
adequate and it is argued that there is no evidence at this stage to support a
polar coordinate model of the type proposed by French, Bryant & Bryant
(1976).

MATERIALS AND METHODS

Fertilized White Leghorn eggs were incubated at 38 °C and windowed on the
third or fourth day of development. The embryos were staged according to
Hamburger & Hamilton (1951) and the eggs were sealed with Sellotape and
returned to the incubator. The embryos were examined at intervals and those
at stages 18 to 21 were used as hosts. A graft site was prepared by excising a
small piece of tissue, about 200 /tm cubed, from the right wing bud opposite
somite 16.
Donor chick embryos were at stages 21-23. In some cases, and in all the
X-irradiation experiments, the donors were quail embryos at stages 21-24.
Quail polarizing regions were used in the X-irradiation experiments because
they are less sensitive to ionizing radiation than those of chick embryos (Smith
et al. 1978; Smith, 1980) but give similar patterns of digits. Pieces of polarizing
region tissue, also about 200 /*m cubed, were transfixed with a platinum pin and
positioned in the host embryos. The window in each egg was sealed with
Sellotape and the egg was returned to the incubator. Some embryos were left
untreated or received a graft of anterior margin tissue. These will be referred to
as 'normal' embryos.

X-irradiation
In the irradiation experiments, embryos were treated through the window in
the egg shell with a Marconi high-voltage X-ray machine set at 230 kV and
15 mA at a range of 30 cm. This gave dose rates of 7-4-8-7 Gy min"1. The total
dose was always 10 Gy. (1 Gy = 100 rad).
 Pattern formation in developing chick wing 129

(e)

Fig. 1. A series of camera-lucida drawings of a wing bud which received an additional

polarizing region at stage 20. (a) 3 h after the graft; (b) 15 h; (c) 23 h; (d) 37 h; (e) 51
h. Notice the increase in width. In (c) the area outlined is that of the progress zone
used in the estimate of the width of buds. The length of the bud is indicated in (d).

Camera-lucida drawings
Camera-lucida drawings of wing buds with polarizing region grafts or of
normal wing buds were made soon after the graft and at various times up to
56 h. A Zeiss camera lucida was attached to a stereo IV b zoom microscope and
the drawings were made at a magnification of x 40.

Histological examination
To study the effects of X-radiation on limb buds, treated and untreated buds
were fixed at various times in half-strength Karnovsky's fixative (Karnovsky,
1965), dehydrated and embedded in Araldite. They were sectioned in a plane
containing the proximodistal and dorsoventral axes at a thickness of 1 /.im and
stained with toluidine blue.
130 J. C. SMITH AND L. WOLPERT

Fig. 2. A series of camera-lucida drawings of a wing bud which received a graft of

anterior margin tissue at stage 20. {a) 2 h after the graft; (b) 17 h; (c) 30 h; (d)
42 h.

Whole mounts
The left and right wings of embryos surviving at 10 days of incubation were
fixed in 5 % trichloroacetic acid, stained with 0-1 % Alcian green 2GX in 1 %
concentrated hydrochloric acid in 70% alcohol, differentiated in acid alcohol,
dehydrated and cleared in methyl salicylate.

Treatment of camera-lucida drawings

The length of each wing bud was defined as the distance from the middle of
the junction of the limb with the body wall to the tip of the primary axis (Fig. 1 d)
The width of a bud was a more difficult parameter to define because this
depends both upon the position along the proximodistal axis and the angle to
this axis at which it is measured. Any measurement that includes the antero-
posterior width of the wing while keeping other variables constant is suitable
and it was decided to measure the area of a 'progress zone' (Summerbell,
Lewis & Wolpert, 1973) at the tip of each limb. This was constructed by drawing
the locus of points 350 /im proximal to the very tip of the limb and then con-
tinuing this line smoothly into the anterior and posterior margins of the bud
(Fig. 1 c). At early stages the area to be measured occupied the whole of the
 Pattern formation in developing chick wing 131

0 8 16 24 32 40 48 56
30 (b)

2-5

? 20

00

0-5

0 8 16 24 32 40 48 56
1819202122 23 24 25 26 27
Time after graft (h) stage
Fig. 3. Graphs of the widths (a) and proximodistal lengths (b) of normal buds and
buds with an additional polarizing region after grafts at stage 18. Solid symbols
( • , • ) : polarizing region grafts; open symbols (O, • ) : normal buds. For the
normal wing buds the least squaies regression line was drawn through all the
points. The regression line through all the polarizing region-grafted points gave an
unsatisfactory fit as judged by examination of the residuals (see Sprent, 1969) and
similarly lines of the form y = aeh and y = a + b (\nx) and polynomials were
unsuitable. However, a good fit was obtained by assuming that the polarizing region
had no effect within the first six hours of the graft and drawing the least squares re*
regression line through all the points later than this. The initial growth in the
anteroposterior axis of the wing buds with a grafted polarizing region was assumed
to be similar to the growth in the normal buds so a line was drawn parallel to the
control line with an intercept on the .y-axis determined by the means of all the
measurements made earlier than 6 h.

dorsal surface of the bud. The areas of the progress zones thus defined were
measured with a planimeter and they will be proportional to the width of the
limb.
Every measurement of length and progress zone area was repeated on a
traced drawing of the limb bud. The length measurements were the more
consistent with a maximum variation about the mean of 1-5% while the
maximum variation in progress zone area was 5%. Both these figures were
132 J. C. SMITH AND L. WOLPERT

1-2
(a)
1= 10
? 0-8
0-6

0-4

0-2

0 8 16 24 32 40 48 56
30
(b)
2-5

2-0

1-5

10

0-5

0 8 16 24 32 40 48 56
21 22 23 24 25 26 27 28
Time after graft (h) stage
Fig. 4. Graphs of widths (a) and proximodistal lengths (Z>) of normal buds and buds
with an additional polarizing region after grafts at stage 21. Solid symbols ( # , • ) :
polarizing region grafts; open symbols (O, • ) : normal buds.

smaller than the variations between embryos, due either to real differences or to
inaccuracies in drawing limb buds or staging. In the results that follow it is the
mean of the two measurements that is presented.

RESULTS
(A) Untreated embryos
At each stage, 18-21, at least five grafted and five normal embryos were
studied. The data are presented in Fig. 3 and 4 as graphs of the widths and lengths
of wing buds with a grafted polarizing region and of normal wing buds plotted
against time. There were no major differences between the four stages examined
so they are discussed together. A typical series of drawings for a wing bud with
a grafted polarizing region is shown in Fig. 1 and for a normal wing bud in
Fig. 2.
(i) The increase in width of wing buds with grafted polarizing region. The widths
of the wing buds with a grafted polarizing region increased dramatically over
 Pattern formation in developing chick wing 133

Table 1. Rates of widening in irradiated and unirradiated

limb buds with a grafted polarizing region
Rate of widening ±95% confidence limits (mm 2 /hx 103)

Irradiated
Bud with grafted bud with grafted
Stage Normal bud polarizing region Irradiated bud polarizing region
18 41 ±0-9 130±l 6 40 ±1-3 8-3 ±4-8
19 4-8 ±0-7 14-6±l-6 2-5±l-8 6-4±3-4
20 3-6±0-7 14-9 ±2-2 20 ±1-5 80±2-4
21 4-7 ±0-9 14-8±l-9 3-8±l-9 8-2±6-l

those of the normal wings (Figs 3a-4). The data at each stage were analysed
in the following way:
The rates at which the normal wing buds and the wing buds with a grafted
polarizing region widened are shown in Table 1. The buds with an additional
polarizing region widened three to four times faster than the normal buds and
Student's Mest showed that there were no significant differences in the rates
of widening between stages, either for normal wing buds or for buds with a
grafted polarizing region. The widths of the buds with a grafted polarizing
region had increased by about 50 % 36 h after the graft in agreement with
Tickle et al. 1975) and by 56 h the widths had approximately doubled.
The times at which the limb buds began to widen were determined by inspec-
tion of the graphs. At all stages a significant widening had occurred by 20 h
after the graft and the increase in width appeared to begin at about 16 h.
(ii) Rates of proximodistal growth. It is possible that the increase in width of
the wing buds with a grafted polarizing region occurred at the expense of their
proximodistal growth. To investigate this the lengths of normal wing buds and
buds with a grafted polarizing region were plotted against time (Figs 3b, Ab).
The least squares regression lines were calculated and the rates of growth are
presented in Table 2. The results at stages 18, 19, 20, 21 are very similar.
Except at stage 18 (see below) the rates of growth of normal wing buds and
of buds with a grafted polarizing region were between 43 and 48 /*m/h. This
range agrees well with a rate calculated from the data of Summerbell (1974a)
of 48 /tm/h. At no stage did the buds with a grafted polarizing region grow
more slowly than the normal buds. Indeed, at stage 18 the growth of the
grafted buds was significantly faster than the normal buds (Student's /-test:
0-002 < P < 0001). It is not clear why this occurred. It appears to be due,
however, to slow growth in the normal buds rather than to accelerated growth
in the buds with a grafted polarizing region. The results, therefore, suggest that
changes in the anteroposterior extent of the limb bud occur independently of
the proximodistal axis.
134 J. C. SMITH AND L. WOLPERT

(a)

Fig. 5. Wings obtained after grafts of an additional polarizing region opposite

somite 16. (a) digit pattern 4 3 2 3 4; (b) digit pattern 4 3 2 2 3 4.

Table 2. Rates of proximodistal growth in irradiated and unirradiated

limb buds with a grafted polarizing region
Rate of growth ±95% confidence limits (jim/ti)
f i

Irradiated
bud with grafted bud with grafted
Stage Normal bud polarizing region Irradiated bud polarizing region
18 37±2 44±6 34±5 35±5
19 43±3 47±3 29±4 33±2
20 48±3 48±3 35±4 30±4
21 46±6 48 ±1 36±2 31±10

The lines drawn on Figs 3b and 4b are the least squares regression lines
combining the data from normal buds and buds with a grafted polarizing
region. The slopes will be compared with the growth rates of limb buds treated
with X-irradiation in the second part of this paper.
(iii) Pattern formation in wing buds with a grafted polarizing region. At least
five normal wing buds and five buds with a grafted polarizing region were
examined at each stage. Only about 35 % of these embryos survived to 10 days
of incubation, less than half the usual number, presumably because the eggs
were removed from the incubator and examined so often. The nine surviving
embryos with a polarizing region graft were fixed, stained with Alcian green
and whole-mounted. Five had the digit pattern 4 3 2 2 3 4 and four 4 3 2 3 4
(Fig. 5). These are similar to the results obtained by Tickle et al. (1975) and
Summerbell & Tickle (1977). All the surviving normal embryos had normal
wings with a digit pattern 2 3 4.

(B) The effect of X-irradiation on growth and pattern formation along the antero-
posterior axis of the limb
Embryos were treated with 10 Gy X-irradiation, as described in the Methods,
within 2 h of a polarizing region graft or a graft of anterior margin tissue. The
 Pattern formation in developing chick wing 135

0-3 mm

Fig. 6. A series of camera-lucida drawings of an irradiated wing bud which received

an additional quail polarizing region at stage 20. (a) 3£ h after the graft; (b) 19 h;
(c)28h;(</)41h;(<?)50h.

embryos were visibly affected by irradiation within about 6 h. Most obviously

damaged were the blood vessels of the yolk sac and the midbrain. Macrophages
could be seen in the limb buds.
At least three and usually four grafted and normal embryos were examined
at each stage.
A typical series of drawings for an irradiated bud with a grafted polarizing
region is shown in Fig. 8 and for an irradiated normal bud in Fig 7. They should
be compared with the drawings in Figs 1 and 2.
(i) The increase in width of irradiated wing buds with grafted polarizing region.
Figures 8 a and 9a show that irradiation severely inhibits the increase in width
of buds with a grafted polarizing region although the widths of irradiated
normal buds differ little from their unirradiated counterparts. Similar results
were obtained for stages 19 and 20. For the normal irradiated buds the least
squares regression lines were drawn. At each stage of operation these lines were
slightly less steep than those for the unirradiated normal limbs (Table 1). The
slopes of the four pairs of lines were compared with a one-tailed Student's
/-test. At two stages, stages 19 and 20, the decrease in slope due to irradiation
was significant (0-005 < P < 0-001, 0-05 < P < 0-02 respectively). However,
136 J. C. SMITH AND L. WOLPERT

0-3 mm

Fig. 7. A series of camera-lucida drawings of an irradiated wing bud which re-

ceived a graft of quail anterior margin tissue at stage 20 {a) 5 h after the graft;
(6) 21 h; (c) 34 h; (i

this effect is not discussed further because it is small compared with the effect of
radiation on widening after a polarizing region graft.
The points obtained for irradiated buds with a grafted polarizing region were
treated in the same way as unirradiated buds with a grafted polarizing region.
The results were very variable, but the rates of widening did not differ signifi-
cantly between stages and all were significantly greater than the rates of widen-
ing of irradiated buds without a polarizing region graft (Student's /-test; see
Table 1). The most interesting observation, however, is that the rates of widening
were significantly lower than after unirradiated polarizing region grafts (Table 1)
This inhibition of widening by X-irradiation is discussed later.
It was not possible to estimate the time at which the irradiated buds with a
grafted polarizing region began to widen because the rates of widening were so
slow.
(ii) Rates of proximodistal growth of irradiated buds. The lengths of the
irradiated buds and of the irradiated buds with a grafted polarizing region are
plotted in Figs 86 and 9 b. The rates of growth of the irradiated buds are
compared in Table 2.
At no stage did the rates of growth of the irradiated buds and the irradiated
buds with a grafted polarizing region differ significantly and it may be concluded
as it was for unirradiated buds, that growth in the anteroposterior axis occurs
independently of the proximodistal axis. The lines drawn on the graphs are the
 Pattern formation in developing chick wing 137
1-2 r
(b)

04
"5
< 0-2

0 8 16 24 32 40 48 56
30
(a)
2-5

^20

£ 1-5
c
- i 10

0-5

0 8 16 24 32 40 .,8 56
1819 20 2122 23 24 25 26 27
Time after graft (h) stage
Fig. 8. Graphs of the widths (a) and proximodistal lengths (b) of irradiated buds
and irradiated buds with an additional polarizing region after grafts at stage 18.
Solid symbols ( # , • ) : polarizing region grafts; open symbols (O, • ) ; controls.
Dashed lines show the rates of widening and proximodistal growth for unirradiated
buds.

least squares regression lines for the combined data from the irradiated buds
and the irradiated buds with a grafted polarizing region.
The irradiated buds always grew more slowly than unirradiated buds (see
Figs 86 and 9b). Similarly, Wolpert, Tickle & Sampford (1979) found that
wing buds treated with 20 Gy X-irradiation grafted to host buds grew more
slowly than controls. They also observed that some recovery of growth occurred
within 48 h but this was not so in these experiments, perhaps because the
whole embryo was treated with radiation.
(iii) Histological study of irradiated wing buds. Irradiated wing buds and
irradiated buds with a grafted polarizing region were fixed in half-strength
Karnovsky's fixative 15,29, 38 and 51 h after irradiation. A series of unirradiated
buds was also fixed. They were embedded in Araldite, sectioned and stained
with toluidine blue. Examples of these sections are shown in Fig. 10.
The normal appearance of the AER at all times after irradiation suggests
138 J. C. SMITH AND L. WOLPERT

1-2

10

0-8

0-6

0-4

0-2

0 8 16 24 32 40 48 56

0 8 16 24 32 40 48 56
21 22 23 24 25 26 27 28
Time after graft (h) stage
Fig. 9. Graphs of the widths (a) and proximodistal lengths (b) of irradiated buds
and irradiated buds with an additional polarizing region after grafts at stage 21.
Solid symbols ( # , • ) : polarizing region grafts; open symbols (O, D) controls.
Dashed lines show the rates of widening and proximodistal growth for unirradiated
buds.

that the inhibition of outgrowth and widening of limbs by X-irradiation was

due to damage to the mesoderm. In their experiments Wolpert et al. (1979)
arrived at a similar conclusion by recombining irradiated mesoderms with
normal ectoderms and vice versa. Most radiation damage was evident at 15 h.
The limb buds contained many macrophages and the cell density at the tip of
the buds was reduced to about 50% of the unirradiated buds (8-8 cells per
1000 jLtm2 compared with 15-8). Mitotic figures were present in both the meso-
derm and the ectoderm (Fig. \0d). No estimate of the mitotic index was made

Fig. 10. The effects of 10 Gy X-irradiation on chick wing buds, (a) An unirradiated
wing bud. (b) High power of (a), (c) 15 h after iiradiation. Notice the reduced cell
density and macrophages. (d) High power of (c): notice mitotic figures (m) and the
apical ectodermal ridge (AER). (e) 29 h after irradiation. There are fewer macro-
phages. (/) High power of (e). (g) 38 h after irradiation, (h) 51 h after irradiation.
Pattern formation in developing chick wing 139

30 urn
140 J. C. SMITH AND L. WOLPERT
(a) (b)

Fig. 11. Wings produced by irradiation of embryos within 2 h of a polarizing region

graft opposite somite 16. (a) digit pattern 4 3 4; (b) digit pattern 4 3 3 4.

but Wolpert et al. (1979) found that the mitotic index in limb buds 12 and 24 h
after treatment with 20 Gy X-irradiation was normal, about 2 %.
By 29 h the irradiated buds were still visibly abnormal but the cell density
at the tip had increased to 11-3 cells per 1000 /on2 and there were fewer macro-
phages. At 38 and 51 h the limbs looked quite normal and the cell densities
at the tips of the buds were at the control levels.
(iv) Pattern formation. Seventeen irradiated embryos with a grafted polarizing
region were used in the examination of the growth of wing buds after irradiation
but only two survived to 10 days of incubation. Therefore, a further 35 embryos
with a grafted polarizing region were allowed to develop after irradiation
without interference, and nine survived. Of these 11 surviving embryos three
had the digit pattern 4 3 4 and six 4 3 3 4 (Fig. 15). There was also one limb
with the pattern 2 2 3 4 and one 3 3 4. In the proximodistal axis of the limbs
there were level-specific abnormalities similar to those described by Summerbell
(1978). For example, the forearm was shortened compared with the digits
(Fig. 11). These results contrast with the results from unirradiated embryos
because digit 2 was not formed in the middle of the reduplicated wings. This is
discussed below.

DISCUSSION
(i) Widening of limb buds after a graft of an additional polarizing region
The results obtained in the first part of this paper indicate that the widening
of a grafted limb bud, one of the earliest responses to an additional polarizing
region, begins about 16 h after the operation, regardless of the stage at which
the graft was performed. This shows that widening may commence at any
stage, in accord with the observation that a reduplication may begin at any
level, depending on the stage of the graft (Summerbell, 1974&). The widening
then proceeds at a rate which produces a 50 % increase in width by 36 h after
the graft and a doubling by about 56 h. The rate of widening does not depend
upon the stage of the graft.
 Pattern formation in developing chick wing 141
The increase in width of wing buds with an additional polarizing region does
not occur at the expense of their proximodistal growth. Therefore, unless there
is a change in the extent of the dorsoventral axis, the polarizing region must
bring about an increase in cell proliferation in the bud. This might occur in two
ways. First, it is known that the apical ectodermal ridge thickens in the anterior
part of the limb bud after a polarizing region graft (Saunders & Gasseling,
1968; Camosso & Roncali, 1968; Smith, 1979a; MacCabe & Parker, 1979).
This might create space for the underlying mesoderm to expand into and the
lowered cell density would bring about an increase in cell division (Summerbell
& Wolpert, 1972). Alternatively, the polarizing region might act directly on the
mesoderm to increase cell division; Camosso & Roncali (1968) claim that the
increase in thickness of the AER following tip rotation occurs after an increase
in the mitotic index of the underlying mesoderm and MacCabe & Parker (1979)
find that the 'memory' (Smith, 19796) of a brief exposure to an additional
polarizing region is retained only by the mesoderm. It is of great interest that
Cooke & Summerbell (1980) have found an enhanced entry to S phase among
mesenchyme cells throughout the progress zone, a few hours after a polarizing
region graft.

(ii) The effect of X-irradiation

X-irradiation produces a decrease in the rate of proximodistal growth of
treated limb buds but it also dramatically reduces the widening of buds that
occurs after a polarizing region graft. This inhibition of widening is probably
due to damage to the mesoderm because histological sections show that this is
quite badly affected by radiation while the ectoderm and AER appear normal.
However, this observation can give no indication as to the cause of widening.
More interestingly, X-irradiation also affects pattern formation. In untreated
buds polarizing region grafts opposite somite 16 gave the digit patterns 4 3 2 3 4
or 4 3 2 2 3 4 (Fig. 5). After X-irradiation the pattern became 4 3 3 4 or even
4 3 4 (Fig. 11). This phenomenon is probably due to a change in the response of
the limb buds rather than to a change in the signal because positional signalling
is quite insensitive to low doses of ionizing radiation (Smith et al. 1978).
One possibility is that X-irradiation reduces the number of cells that are
available to contribute to structures in the anteroposterior axis of the wing.
If this is so, then which digits form will depend upon the threshold number of
cells required for the development of each digit (Wolpert et al. 1979). On this
view, digit 2 is indeed the most sensitive to X-irradiation (Wolpert et al. 1979)
but doses of only 10 Gy prior to stage 24 are insufficient to affects its develop-
ment. Furthermore, digit 4 is more sensitive to radiation than digit 3, so the
production of the digit pattern 4 3 4 cannot be explained in this way.
Another suggestion might be that the observed reduction in cell density after
X-irradiation, changes the properties of the responding mesoderm so as to
make a morphogen concentration profile less steep and so the positional values
142 J. C. SMITH AND L. WOLPERT

25% 50%

0-6 0-8 10 1-2 1-4

Distance (mm)
Fig. 12. The effect of widening on the concentration profile of a morphogen pro-
duced by the host polarizing region and a polarizing region grafted opposite somite
16. Three profiles are shown for 0, 25 and 50% widening. It is assumed that the
polarizing region holds the concentration of the diffusible morphogen at 100 and that
it is degraded at a rate proportional to its concentration. The threshold concentra-
tion for each digit was chosen to be in line with SummerbeH's fatemap (1979) for the
digits (Wolpert & Hornbruch, 1981). It can be seen that if no widening occurs the
concentration of the morphogen is too high for digit 2 to form. Digit 2 may form
with 25 % widening.

in the middle of the limb higher. This could occur if, for example, the rate of
destruction of the morphogen was reduced without changing the diffusion con-
stant for the passage of the morphogen through the limb.
However, the simplest and most attractive explanation is the one mentioned
in the Introduction to this paper that the inhibition of widening prevented the
concentration of morphogen in the middle of the limb falling to a level that
would specify digit 2. That is, it prevented 'distal deepening' (Slack, 1977).
Normally a polarizing region graft brings about a 50 % increase in width by
stage 24 or 25, when the digits are being laid down (Figs la and 4a; Tickle
et al. 1975). Irradiation inhibits widening such that, at the same time after the
graft, the limb bud is only about 15 % wider (Figs 8 a and 9 a). The change in
the morphogen concentration profile that might result is illustrated in Fig. 12.
This interpretation is strengthened by some recent experiments of Hornbruch
& Wolpert (unpublished). Embryos with grafts of chick polarizing regions were
treated with 10 Gy X-irradiation at various times after the operation. When
irradiation was delayed until 18 h after the operation, by which time some
widening had occurred, half the resulting reduplicated wings did contain
digit 2. When irradiation was delayed until 28 or 42 h a typical reduplicated
wing was rarely formed but the host digit 2 was intact. It is possible that the
rapidly-growing reduplicated structures are particularly sensitive to X-irradia-
tion (see for example, Coggle, 1973).
 Pattern formation in developing chick wing 143

We thank Dr C. Tickle for her comments, Lynne Dillon for typing the manuscript
and the MRC for financial support.

REFERENCES
CAMOSSO, M. & RONCALI, L. (1968). Time sequence of the process of ectodermal ridge
thickening and of mesodermal cell proliferation during apical outgrowth of the chick
embryo limb bud. Acta Embryol. Morph. exp. 10, 247-263.
COGGLE, J. E. (1973). Biological Effects of Radiation. London and Winchester: Wykeham
Publications (London) Ltd.
COOKE, J. & SUMMERBELL, D. (1980). Cell cycle and experimental pattern duplication in the
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(Received 1 July 1980, revised 15 December 1980)