/ . Embryol. exp. Morph. Vol. 60, pp. 321-328, 1980
Printed in Great Britain © Company of Biologists Limited 1980
321
The time required for positional signalling in
the chick wing bud
By J. C. SMITH 1
From the Department of Biology as Applied to Medicine, The Middlesex Hospital
Medical School, London
SUMMARY
An estimate of the time required for positional signalling in the chick wing bud was
obtained by grafting irradiated quail polarizing regions to host wing buds and removing them
at various times. Such polarizing regions must be present for at least 15 h to induce additional
structures in the host wings. This result is discussed in terms of a diffusible morphogen model.
INTRODUCTION
The polarizing region is a small group of cells at the posterior margin of the
developing chick limb 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). Although Saunders
(1977) argues that the polarizing region is not active in normal development
there is reason to believe that it is responsible for establishing positional information (Wolpert, 1969, 1971) along the antero-posterior axis of the limb bud
(Summerbell, 1979; Smith, 1979).
One particular model for the establishment of positional information suggests
that the polarizing region acts by producing a diffusible morphogen, the
concentration of which falls as distance from the polarizing region increases
(Wolpert, 1969; Tickle et al 1975; Summerbell & Tickle, 1977; Smith, Tickle &
Wolpert, 1978). There is some evidence for the existence of such a morphogen
(MacCabe & Parker, 1975,1976; MacCabe, Calandra & Parker, 1977; Calandra
& MacCabe, 1978). The responding cells are assumed to be able to interpret the
concentration of morphogen and so behave according to their position. For
instance, digit 4 forms closest to the polarizing region where the concentration
of morphogen would be highest, then digit 3, then digit 2. The responding cells
may be able to 'remember' the highest concentration of morphogen they have
ever experienced (Smith, 1979).
1
Author's address: Laboratory of Tumor Biology, Group W, The Sidney Farber Cancer
Institute, 44Binney Street, Boston, MA. 02115, U.S.A.
322
J. C. SMITH
In this paper an estimate is made of the time that is required by the polarizing
region to respecify positional information along the antero-posterior axis of the
chick wing bud. This time is a useful one to know for at least two reasons. First,
it places restrictions on the models that may be proposed for the action of the
polarizing region. If, for example, the polarizing region could act in a matter of
minutes, then the model based on the diffusion of a morphogen would be extremely unlikely (Wolpert, 1971). Secondly, the time needed for positional
signalling is a requirement for experiments in which attempts are made to mimic
the effect of the polarizing region.
In previous efforts to estimate the time required for positional signalling,
responding tissue has been exposed to polarizing tissue for various times. This
was done either by re-orienting the tip of a wing bud which had been rotated
through 180° (Saunders & Gasseling, 1963) or by interposing a tantalum foil
barrier between a grafted polarizing region and the host tissue (Summerbell,
1973). The former method suggested that 12 h is required for signalling; the
latter 10 h. Both these times are consistent with the idea that positional information is specified by a diffusible morphogen, but neither technique was able to
exclude the possibility that some polarizing region tissue had been left in contact
with responding tissue after it was presumed to have been removed. It is possible
that the time of 12 h or so thought to be required for signalling is simply the
time it takes for polarizing region cells to spread along the limb in sufficient
numbers to signal when the main body of the graft is removed.
Here, an attempt is made to overcome this difficulty by grafting quail
polarizing regions treated with 100 Gy y-radiation. Such polarizing regions are
capable of producing complete reduplications in host wings (Smith et al. 1978)
and they offer several advantages over untreated polarizing regions. They can
be recognized as white areas in the host wings (Smith, 1979); the cells do not
divide for at least 12 h after the graft (Smith, in preparation); and it is known
that their signalling ability is not passed on to adjacent tissue (Smith, 1979).
It should be much easier, therefore, to remove all of an irradiated polarizing
region from a wing bud and so gain another estimate of the time needed for
positional signalling.
MATERIALS AND METHODS
Fertilized White Leghorn eggs were incubated at 38 °C and windowed on the
third 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 19-21
were used as hosts. A graft site was prepared by excising a small piece of tissue,
about 200 [im cubed, from the right wing bud opposite somite 16.
Quail embryos at stages 21-24 were treated in ovo with 100 Gy y-radiation as
described previously (Smith et al. 1978) and pieces of polarizing region tissue,
also about 200 jam cubed, were transfixed with a platinum pin and positioned in
Time required for positional signalling
(a)
323
(b)
0-3 mm
Fig. .1. Camera-lucida drawings to show the removal of an irradiated quail polarizing region from a chick wing bud. (a) Immediately after the graft; (b) removal of the
polarizing region 12 h later. Notice that some of the surrounding mesoderm is also
removed.
the host embryos. These embryos were returned to the incubator and at various
times the grafted tissue, with some of the surrounding mesoderm, was cut out of
the host wing bud and discarded. The eggs were reincubated and at 10 days of
incubation the left and right wings of the embryos were removed. They 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. Camera-lucida drawings were
sometimes made at the time of grafting and at the time at which the grafted
polarizing region was removed.
In four cases the limb from which the polarizing region had been removed
was fixed about 15 min later in half-strength Karnovsky's fixative (Karnovsky,
1965), dehydrated, and embedded in Araldite. Serial 1|- jam sections were cut
in a plane containing the antero-posterior and proximo-distal axes and alternate
sections were stained with toluidine blue or by the Feulgen technique. Quail
cells may be recognized by their distinctive Feulgen-positive nucleoli (LeDouarin, 1973) and these sections were to check that all of the grafted polarizing
region could be removed.
RESULTS
It was usually possible to recognize the grafted tissue for up to 24 h after the
operation. In six cases it could not be seen clearly and these embryos were
allowed to develop without interference; the results are not considered here.
In the remainder, the grafted polarizing region was removed 4,12,15,17 or 24 h
after the operation. Figure 1 shows camera-lucida drawings of a wing bud at
the time of the graft and when the polarizing region was removed 12 h later. It
illustrates the amount of host tissue that was usually removed with the polarizing
324
J. C. SMITH
Table 1. The time required for positional signalling
Time polarizing region
removed (h)
Pattern of digits
3
2
3
2
2
2
2
2
3
3
3
3
4
4
4
4
4
12
15
17
7
1
7
1
2
4
1
1
6
1
2
24
1
3
1
3*
* Identification of one of these was equivocal.
i L
Fig. 2. Typical wings obtained after removing polarizing regions at different times.
(a) Polarizing region removed after 12 h; digit pattern 2 3 4. (b) Polarizing region
removed after 15 h; digit pattern 2 2 3 4. (c) Polarizing region removed after 17 h;
digit pattern 3 2 2 3 4. In (b) and (c) the radius is absent. This was observed quite
frequently in those limbs with an additional digit 2 or 3 (18 out of 23) but not in
normal wings (one out of 19). The reason for this is unclear.
Time required for positional signalling
325
ISC
«!»&W
*£^S .••:'-•• rtZMFig. 3. A histological section, stained by the Feulgen technique, of a limb from
which a grafted irradiated polarizing region was presumed to have been removed
.15 min before fixation. Some quail cells remain. The quail nucleolar marker is
indistinct because the cells were irradiated, (a) Scale bar is 100/tm. (b) The area
outlined in (a). Scale bar is 25 /im. Quail cells (Q) can be recognized by a distinctive
Feulgen-positive nucleolus.
region. The embryos were then re-incubated until day 10 of incubation when the
wings were fixed, stained and whole-mounted.
The results are presented in Table 1 and illustrated in Fig. 2. In the Table, the
extent of duplication is indicated by the pattern of digits. A normal wing has the
digit pattern 2 3 4, and a fully reduplicated wing the pattern 4 3 22 34;
intermediate duplications will lack the additional digit 4 and perhaps digit 3.
Removal of the polarizing region at 4 or 12 h almost always resulted in normal
wings (Fig. 2a). At both times there was one limb out of eight with the digit
pattern 2 2 3 4. At other times more structures were formed the later the
326
J. C. SMITH
polarizing region was removed. At 15 h four limbs out of seven contained an
extra digit 2 and one limb contained the extra digits 2 and 3. By 17 h six limbs
out of ten contained an extra digit 2 and three an extra digit 3 and at 24 h four
limbs out of eight contained an extra digit 3 and three an extra digit 2. Wings
with an additional digit 4 were never formed.
Four limbs were fixed about 15 min after excision of the grafted polarizing
region, one 15 h and three 17 h after the graft. In sections of three of these there
were no quail cells but in one, from which the polarizing region was removed
17 h after the graft, a small group of grafted cells remained (Fig. 3). These cells
could be recognized by their Feulgen-positive nucleoli, although these were
faint compared with those of unirradiated quail polarizing region cells (see also
Smith, 1979).
DISCUSSION
Any method for determining the time required for positional signalling that
involves isolating a grafted polarizing region from the responding tissue must
strike a compromise between making sure that all of the grafted tissue has been
removed and trying to avoid removing any of the responding tissue. It was
intended that use of irradiated polarizing regions would make it easier to
remove all of the graft without having to take too much of the surrounding
mesoderm as well. The amount of host mesoderm that was usually removed is
illustrated in Fig. 1. In three of the four cases when the operated limb bud was
examined histologically, no quail cells were seen; furthermore, the results in
Table 1 are quite consistent and this suggests that in most of the experiments
the graft was indeed removed completely.
When the polarizing region was removed 15 h after the operation an additional
digit 2 was frequently formed and after 17 h digits 2 and 3. Some of the host
mesoderm that was removed with the polarizing region probably included
specified tissue and so these results must be regarded as underestimates of the
total amount of tissue that had been specified. Allowing for this, about 15 h is a
reasonable estimate for the time required to respecify positional information.
This time of 15 h would appear, like the 10 and 12 h of Summerbell (1973) and
Saunders & Gasseling (1963), to be consistent with the diffusible morphogen
model for the specification of positional information although it cannot, of
course, rule out any other mechanisms, perhaps based on local interactions
(Iten & Murphy, 1980). However, it is worth discussing further the time course
of events in a model based on the diffusion of a morphogen because in such a
model the measured time required for signalling is made up of three components:
first, the time needed for the grafted polarizing region to heal in and begin
signalling; second, the time needed to setup a gradient of positional information;
and third, the time taken by the responding cells to change their positional
values. These stages need not happen strictly sequentially; cells may be changing
their positional values before the gradient of positional information has reached
equilibrium. It is of great interest to know the contribution of each of these
Time required for positional signalling
327
components to the total time, for if the second component is very short, the
diffusible morphogen model would be very unlikely.
The first phase, in which the polarizing region heals in to the limb bud before
beginning to signal, may be short compared with the 15 h. If cell-to-cell contact
is not required for signalling (see Saunders & Gasseling, 1963; MacCabe &
Parker, 1975) there is no reason why signalling should not begin almost at once.
If cell contact is needed it may take longer, but it is known, for example, that
low resistance junctions between newly apposed sponge (Loewenstein, 1967) or
newt-embryo (Ito & Loewenstein, 1969) cells can form within minutes. It is
possible that the polarizing region signals through such junctions (Furshpan &
Potter, 1968; Wolpert, 1978).
By contrast, changes in positional values may take some hours; it has been
suggested, for example, that positional values can only change with cell division
(Lawrence, Crick & Munro, 1972; Meinhardt, 1977). Values obtained for the
cell cycle time in the chick wing bud vary. Cairns (1966) has estimated a figure
of 7 h at the relevant stages whereas Janners & Searls (1970) find the time to be
about 13 h. Summerbell & Lewis (1975) and Lewis (1975) suggest that this
latter figure is an overestimate based on underestimates of labelling index. Thus,
if a figure of 7 h is accepted as reasonable, about half of the 15 h required for
signalling might be devoted to establishing a gradient of positional information.
This time is still, however, consistent with the diffusible morphogen model.
For the insect egg, Meinhardt (1977) has proposed that cells proceed to their
final positional values, as defined by the local morphogen concentration, step by
step. In the chick wing bud this would mean that a cell with the positional value
for, say, digit 4 will have passed through the positional values appropriate to
digits 2 and 3. This might explain why digits with higher positional values are
formed with increasing times of exposure topolarizing region tissue (see Table 1).
Digit 4 was never formed in these experiments; possibly because it forms closest
to the polarizing region and was always excised but perhaps because the polarizing region tissue was not left in place long enough.
I thank Larry Honig, Richard Swaab, Cheryll Tickle and Lewis Wolpert for helpful
comments, and Lynne Dillon for typing the manuscript. I also thank the Medical Research
Council for a research studentship.
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{Received 11 March 1980, revised 12 May 1980)