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.

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 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.

One of the earliest responses to a polarizing region graft is an increase in the 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 experiments 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).

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 μ m 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 μ 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).

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 × 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 μm and stained with toluidine blue.

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)

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).

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).

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 anteroposterior 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 μm proximal to the very tip of the limb and then continuing 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 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 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.

(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.

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.

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.

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 (◯, □): normal buds. For the normal wing buds the least squates 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 = aeb and y = a + b(Inx) 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.

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 (◯, □): normal buds. For the normal wing buds the least squates 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 = aeb and y = a + b(Inx) 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.

Fig. 4

Graphs of widths (a) and proximodistal lengths (b) of normal buds and buds with an additional polarizing region after grafts at stage 21. Solid symbols (●>◼): polarizing region grafts; open symbols (◯, □): normal buds.

Fig. 4

Graphs of widths (a) and proximodistal lengths (b) of normal buds and buds with an additional polarizing region after grafts at stage 21. Solid symbols (●>◼): polarizing region grafts; open symbols (◯, □): normal buds.

(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 those of the normal wings (Figs 3a4). 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 /-test 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.

Table 1

Rates of widening in irradiated and unirradiated limb buds with a grafted polarizing region

Rates of widening in irradiated and unirradiated limb buds with a grafted polarizing region
Rates of widening in irradiated and unirradiated limb buds with a grafted polarizing region

The times at which the limb buds began to widen were determined by inspection 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 3 b, 4b). 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.

Table 2

Rates of proximodistal growth in irradiated and unirradiated limb buds with a grafted polarizing region

Rates of proximodistal growth in irradiated and unirradiated limb buds with a grafted polarizing region
Rates of proximodistal growth in irradiated and unirradiated limb buds with a grafted polarizing region

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 μm/h. This range agrees well with a rate calculated from the data of Summerbell (1974a) of 48 μm/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 t-test: 0·002 < P < 0·001). 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.

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.

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.

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.

Fig. 6

A series of camera-lucida drawings of an irradiated wing bud which received an additional quail polarizing region at stage 20. (a)312 h after the graft; (b) 19 h; (c) 28 h; (d)41 h; (e) 50 h.

Fig. 6

A series of camera-lucida drawings of an irradiated wing bud which received an additional quail polarizing region at stage 20. (a)312 h after the graft; (b) 19 h; (c) 28 h; (d)41 h; (e) 50 h.

(B) The effect of X-irradiation on growth and pattern formation along the anteroposterior 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 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.

Fig. 7

A series of camera-lucida drawings of an irradiated wing bud which received a graft of quail anterior margin tissue at stage 20 (a) 5 h after the graft; (b) 21 h; (c) 34 h; (d) 44 h.

Fig. 7

A series of camera-lucida drawings of an irradiated wing bud which received a graft of quail anterior margin tissue at stage 20 (a) 5 h after the graft; (b) 21 h; (c) 34 h; (d) 44 h.

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 (◯, □); controls. Dashed lines show the rates of widening and proximodistal growth for unirradiated buds.

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 (◯, □); controls. Dashed lines show the rates of widening and proximodistal growth for unirradiated buds.

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 (◯, □) controls. Dashed lines show the rates of widening and proximodistal growth for unirradiated buds.

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 (◯, □) controls. Dashed lines show the rates of widening and proximodistal growth for unirradiated buds.

(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 t-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, 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 significantly between stages and all were significantly greater than the rates of widening of irradiated buds without a polarizing region graft (Student’s t-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 8a and 9b. 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 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 8 b and 9 b). 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.

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. (J) High power of (c): notice mitotic figures (m) and the apical ectodermal ridge (AER). (e) 29 h after irradiation. There are fewer macrophages. (f) High power of (e). (g) 38 h after irradiation. (h) 51 h after irradiation.

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. (J) High power of (c): notice mitotic figures (m) and the apical ectodermal ridge (AER). (e) 29 h after irradiation. There are fewer macrophages. (f) High power of (e). (g) 38 h after irradiation. (h) 51 h after irradiation.

The normal appearance of the AER at all times after irradiation suggests 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 μ m2 compared with 15·8). Mitotic figures were present in both the mesoderm and the ectoderm (Fig. 10 d). No estimate of the mitotic index was made 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 /un2 and there were fewer macrophages. 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.

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.

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.

(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 b). 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.

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, 1979b) 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 development. 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 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 constant 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 3 a 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 8a and 9a). The change in the morphogen concentration profile that might result is illustrated in Fig. 12.

Fig. 12

The effect of widening on the concentration profile of a morphogen produced 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 concentration for each digit was chosen to be in line with Summerbell’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.

Fig. 12

The effect of widening on the concentration profile of a morphogen produced 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 concentration for each digit was chosen to be in line with Summerbell’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.

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-irradiation (see for example, Coggle, 1973).

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

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