/. Embryol exp. Morph. Vol. 63, pp. 127-144, 1981 127Printed in Great Britain © Company of Biologists Limited 1981

Pattern formation along the anteroposterioraxis of the chick wing: the increase in widthfollowing a polarizing region graft and the

effect of X-irradiation

By J. C. SMITH1 AND L. WOLPERT2

From the Department of Biology as Applied to Medicine, The Middlesex HospitalMedical School, London

SUMMARY

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

INTRODUCTION

Positional information along the anteroposterior axis of the chick wing budappears to be specified by the polarizing region at the posterior margin of thebud. When tissue from this region is grafted to the anterior border of a bud, alimb 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 considerableamount of evidence suggests that the polarizing region acts by producing adiffusible morphogen, the concentration of which is highest close to thepolarizing 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 instancedigit 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 the1 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 importancein the specification of positional information for without it the concentrationof morphogen in the middle of a host wing would not fall low enough to specifydigit 2. In the first part of this paper a study is made of the increase in widthof wing buds to which an additional polarizing region has been grafted. Inthe second part it is found that the widening is greatly inhibited by a low doseof X-irradiation and the pattern of digits that results is consistent with thediffusible morphogen model since digit 2 is absent.

It should be noted that Iten & Murphy (1980) have suggested that the patternof digits following polarizing region grafts may be due to intercalation involvingepimorphosis. This is discussed by Wolpert & Hornbruch (1981) where experi-ments are presented which suggest that a linear intercalation model is notadequate and it is argued that there is no evidence at this stage to support apolar 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 thethird or fourth day of development. The embryos were staged according toHamburger & Hamilton (1951) and the eggs were sealed with Sellotape andreturned to the incubator. The embryos were examined at intervals and thoseat stages 18 to 21 were used as hosts. A graft site was prepared by excising asmall piece of tissue, about 200 /tm cubed, from the right wing bud oppositesomite 16.

Donor chick embryos were at stages 21-23. In some cases, and in all theX-irradiation experiments, the donors were quail embryos at stages 21-24.Quail polarizing regions were used in the X-irradiation experiments becausethey are less sensitive to ionizing radiation than those of chick embryos (Smithet al. 1978; Smith, 1980) but give similar patterns of digits. Pieces of polarizingregion tissue, also about 200 /*m cubed, were transfixed with a platinum pin andpositioned in the host embryos. The window in each egg was sealed withSellotape and the egg was returned to the incubator. Some embryos were leftuntreated or received a graft of anterior margin tissue. These will be referred toas 'normal' embryos.

X-irradiation

In the irradiation experiments, embryos were treated through the window inthe egg shell with a Marconi high-voltage X-ray machine set at 230 kV and15 mA at a range of 30 cm. This gave dose rates of 7-4-8-7 Gy min"1. The totaldose 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 additionalpolarizing region at stage 20. (a) 3 h after the graft; (b) 15 h; (c) 23 h; (d) 37 h; (e) 51h. Notice the increase in width. In (c) the area outlined is that of the progress zoneused 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 ofnormal wing buds were made soon after the graft and at various times up to56 h. A Zeiss camera lucida was attached to a stereo IV b zoom microscope andthe drawings were made at a magnification of x 40.

Histological examination

To study the effects of X-radiation on limb buds, treated and untreated budswere fixed at various times in half-strength Karnovsky's fixative (Karnovsky,1965), dehydrated and embedded in Araldite. They were sectioned in a planecontaining the proximodistal and dorsoventral axes at a thickness of 1 /.im andstained 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 ofanterior 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 werefixed 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 ofthe 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 thisdepends both upon the position along the proximodistal axis and the angle tothis axis at which it is measured. Any measurement that includes the antero-posterior width of the wing while keeping other variables constant is suitableand 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 drawingthe 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

30

2-5

? 20

00

0-5

0 8 16 24 32 40 48 56

(b)

0 8 16 24 32 40 48 561819202122 23 24 25 26 27

Time after graft (h) stage

Fig. 3. Graphs of the widths (a) and proximodistal lengths (b) of normal buds andbuds with an additional polarizing region after grafts at stage 18. Solid symbols( • , • ) : polarizing region grafts; open symbols (O, • ) : normal buds. For thenormal wing buds the least squaies regression line was drawn through all thepoints. The regression line through all the polarizing region-grafted points gave anunsatisfactory fit as judged by examination of the residuals (see Sprent, 1969) andsimilarly lines of the form y = aeh and y = a + b (\nx) and polynomials wereunsuitable. However, a good fit was obtained by assuming that the polarizing regionhad 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 theanteroposterior axis of the wing buds with a grafted polarizing region was assumedto be similar to the growth in the normal buds so a line was drawn parallel to thecontrol line with an intercept on the .y-axis determined by the means of all themeasurements made earlier than 6 h.

dorsal surface of the bud. The areas of the progress zones thus defined weremeasured with a planimeter and they will be proportional to the width of thelimb.

Every measurement of length and progress zone area was repeated on atraced drawing of the limb bud. The length measurements were the moreconsistent with a maximum variation about the mean of 1-5% while themaximum variation in progress zone area was 5%. Both these figures were

132 J. C. SMITH AND L. WOLPERT

1-2

1= 10

? 0-8

0-6

0-4

0-2

30

2-5

2-0

1-5

10

0-5

(a)

0 8 16 24 32 40 48 56

(b)

0 8 16 24 32 40 48 5621 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 budswith 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 toinaccuracies in drawing limb buds or staging. In the results that follow it is themean of the two measurements that is presented.

RESULTS

(A) Untreated embryos

At each stage, 18-21, at least five grafted and five normal embryos werestudied. The data are presented in Fig. 3 and 4 as graphs of the widths and lengthsof wing buds with a grafted polarizing region and of normal wing buds plottedagainst time. There were no major differences between the four stages examinedso they are discussed together. A typical series of drawings for a wing bud witha grafted polarizing region is shown in Fig. 1 and for a normal wing bud inFig. 2.

(i) The increase in width of wing buds with grafted polarizing region. The widthsof 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 unirradiatedlimb buds with a grafted polarizing region

Rate of widening ±95% confidence limits (mm2/hx 103)

IrradiatedBud with grafted bud with grafted

Stage Normal bud polarizing region Irradiated bud polarizing region

18192021

41 ±0-94-8 ±0-73-6±0-74-7 ±0-9

130±l 614-6±l-614-9 ±2-214-8±l-9

40 ±1-32-5±l-820 ±1-53-8±l-9

8-3 ±4-86-4±3-480±2-48-2±6-l

those of the normal wings (Figs 3a-4). The data at each stage were analysedin the following way:

The rates at which the normal wing buds and the wing buds with a graftedpolarizing region widened are shown in Table 1. The buds with an additionalpolarizing region widened three to four times faster than the normal buds andStudent's Mest showed that there were no significant differences in the ratesof widening between stages, either for normal wing buds or for buds with agrafted polarizing region. The widths of the buds with a grafted polarizingregion had increased by about 50 % 36 h after the graft in agreement withTickle 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 hafter 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 ofthe wing buds with a grafted polarizing region occurred at the expense of theirproximodistal growth. To investigate this the lengths of normal wing buds andbuds with a grafted polarizing region were plotted against time (Figs 3b, Ab).The least squares regression lines were calculated and the rates of growth arepresented 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 andof buds with a grafted polarizing region were between 43 and 48 /*m/h. Thisrange 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 growmore slowly than the normal buds. Indeed, at stage 18 the growth of thegrafted 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 growthin the buds with a grafted polarizing region. The results, therefore, suggest thatchanges in the anteroposterior extent of the limb bud occur independently ofthe proximodistal axis.

134 J. C. SMITH AND L. WOLPERT

(a)

Fig. 5. Wings obtained after grafts of an additional polarizing region oppositesomite 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 unirradiatedlimb buds with a grafted polarizing region

Rate of growth ±95% confidence limits (jim/ti)f i

Irradiatedbud with grafted bud with grafted

Stage Normal bud polarizing region Irradiated bud polarizing region

18192021

37±243±348±346±6

44±647±348±348 ±1

34±529±435±436±2

35±533±230±431±10

The lines drawn on Figs 3b and 4b are the least squares regression linescombining the data from normal buds and buds with a grafted polarizingregion. The slopes will be compared with the growth rates of limb buds treatedwith X-irradiation in the second part of this paper.

(iii) Pattern formation in wing buds with a grafted polarizing region. At leastfive normal wing buds and five buds with a grafted polarizing region wereexamined at each stage. Only about 35 % of these embryos survived to 10 daysof incubation, less than half the usual number, presumably because the eggswere removed from the incubator and examined so often. The nine survivingembryos with a polarizing region graft were fixed, stained with Alcian greenand 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) andSummerbell & Tickle (1977). All the surviving normal embryos had normalwings 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 receivedan 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 obviouslydamaged were the blood vessels of the yolk sac and the midbrain. Macrophagescould be seen in the limb buds.

At least three and usually four grafted and normal embryos were examinedat each stage.

A typical series of drawings for an irradiated bud with a grafted polarizingregion is shown in Fig. 8 and for an irradiated normal bud in Fig 7. They shouldbe 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 widthof buds with a grafted polarizing region although the widths of irradiatednormal buds differ little from their unirradiated counterparts. Similar resultswere obtained for stages 19 and 20. For the normal irradiated buds the leastsquares regression lines were drawn. At each stage of operation these lines wereslightly less steep than those for the unirradiated normal limbs (Table 1). Theslopes 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 irradiationwas 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 ofradiation on widening after a polarizing region graft.

The points obtained for irradiated buds with a grafted polarizing region weretreated 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; seeTable 1). The most interesting observation, however, is that the rates of wideningwere 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 agrafted polarizing region began to widen because the rates of widening were soslow.

(ii) Rates of proximodistal growth of irradiated buds. The lengths of theirradiated buds and of the irradiated buds with a grafted polarizing region areplotted in Figs 86 and 9 b. The rates of growth of the irradiated buds arecompared in Table 2.

At no stage did the rates of growth of the irradiated buds and the irradiatedbuds with a grafted polarizing region differ significantly and it may be concludedas it was for unirradiated buds, that growth in the anteroposterior axis occursindependently of the proximodistal axis. The lines drawn on the graphs are the

Pattern formation in developing chick wing 137

1-2 r

"5 0 4

< 0-2

30

2-5

^ 2 0

£ 1-5c

-i 10

0-5

(b)

0 8 16 24 32 40 48 56

(a)

0 8 16 24 32 40 .,8 561819 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 budsand 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 unirradiatedbuds.

least squares regression lines for the combined data from the irradiated budsand the irradiated buds with a grafted polarizing region.

The irradiated buds always grew more slowly than unirradiated buds (seeFigs 86 and 9b). Similarly, Wolpert, Tickle & Sampford (1979) found thatwing buds treated with 20 Gy X-irradiation grafted to host buds grew moreslowly than controls. They also observed that some recovery of growth occurredwithin 48 h but this was not so in these experiments, perhaps because thewhole embryo was treated with radiation.

(iii) Histological study of irradiated wing buds. Irradiated wing buds andirradiated buds with a grafted polarizing region were fixed in half-strengthKarnovsky's fixative 15,29, 38 and 51 h after irradiation. A series of unirradiatedbuds was also fixed. They were embedded in Araldite, sectioned and stainedwith 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 5621 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 budsand 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 unirradiatedbuds.

that the inhibition of outgrowth and widening of limbs by X-irradiation wasdue to damage to the mesoderm. In their experiments Wolpert et al. (1979)arrived at a similar conclusion by recombining irradiated mesoderms withnormal 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 ofthe buds was reduced to about 50% of the unirradiated buds (8-8 cells per1000 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 unirradiatedwing bud. (b) High power of (a), (c) 15 h after iiradiation. Notice the reduced celldensity and macrophages. (d) High power of (c): notice mitotic figures (m) and theapical 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 regiongraft 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 hafter treatment with 20 Gy X-irradiation was normal, about 2 %.

By 29 h the irradiated buds were still visibly abnormal but the cell densityat 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 densitiesat the tips of the buds were at the control levels.

(iv) Pattern formation. Seventeen irradiated embryos with a grafted polarizingregion were used in the examination of the growth of wing buds after irradiationbut only two survived to 10 days of incubation. Therefore, a further 35 embryoswith a grafted polarizing region were allowed to develop after irradiationwithout interference, and nine survived. Of these 11 surviving embryos threehad the digit pattern 4 3 4 and six 4 3 3 4 (Fig. 15). There was also one limbwith the pattern 2 2 3 4 and one 3 3 4. In the proximodistal axis of the limbsthere 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 embryosbecause digit 2 was not formed in the middle of the reduplicated wings. This isdiscussed below.

DISCUSSION

(i) Widening of limb buds after a graft of an additional polarizing regionThe 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 polarizingregion, begins about 16 h after the operation, regardless of the stage at whichthe graft was performed. This shows that widening may commence at anystage, in accord with the observation that a reduplication may begin at anylevel, depending on the stage of the graft (Summerbell, 1974&). The wideningthen proceeds at a rate which produces a 50 % increase in width by 36 h afterthe graft and a doubling by about 56 h. The rate of widening does not dependupon the stage of the graft.

Pattern formation in developing chick wing 141

The increase in width of wing buds with an additional polarizing region doesnot occur at the expense of their proximodistal growth. Therefore, unless thereis a change in the extent of the dorsoventral axis, the polarizing region mustbring about an increase in cell proliferation in the bud. This might occur in twoways. First, it is known that the apical ectodermal ridge thickens in the anteriorpart 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 thelowered cell density would bring about an increase in cell division (Summerbell& Wolpert, 1972). Alternatively, the polarizing region might act directly on themesoderm to increase cell division; Camosso & Roncali (1968) claim that theincrease in thickness of the AER following tip rotation occurs after an increasein the mitotic index of the underlying mesoderm and MacCabe & Parker (1979)find that the 'memory' (Smith, 19796) of a brief exposure to an additionalpolarizing region is retained only by the mesoderm. It is of great interest thatCooke & Summerbell (1980) have found an enhanced entry to S phase amongmesenchyme cells throughout the progress zone, a few hours after a polarizingregion graft.

(ii) The effect of X-irradiation

X-irradiation produces a decrease in the rate of proximodistal growth oftreated limb buds but it also dramatically reduces the widening of buds thatoccurs after a polarizing region graft. This inhibition of widening is probablydue to damage to the mesoderm because histological sections show that this isquite 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 untreatedbuds polarizing region grafts opposite somite 16 gave the digit patterns 4 3 2 3 4or 4 3 2 2 3 4 (Fig. 5). After X-irradiation the pattern became 4 3 3 4 or even4 3 4 (Fig. 11). This phenomenon is probably due to a change in the response ofthe limb buds rather than to a change in the signal because positional signallingis quite insensitive to low doses of ionizing radiation (Smith et al. 1978).

One possibility is that X-irradiation reduces the number of cells that areavailable 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 ofcells required for the development of each digit (Wolpert et al. 1979). On thisview, 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 theproduction of the digit pattern 4 3 4 cannot be explained in this way.

Another suggestion might be that the observed reduction in cell density afterX-irradiation, changes the properties of the responding mesoderm so as tomake a morphogen concentration profile less steep and so the positional values

142 J. C. SMITH AND L. WOLPERT

25% 50%

0-6 0-8Distance (mm)

10 1-2 1-4

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 somite16. Three profiles are shown for 0, 25 and 50% widening. It is assumed that thepolarizing region holds the concentration of the diffusible morphogen at 100 and thatit 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 thedigits (Wolpert & Hornbruch, 1981). It can be seen that if no widening occurs theconcentration of the morphogen is too high for digit 2 to form. Digit 2 may formwith 25 % widening.

in the middle of the limb higher. This could occur if, for example, the rate ofdestruction 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 mentionedin the Introduction to this paper that the inhibition of widening prevented theconcentration of morphogen in the middle of the limb falling to a level thatwould specify digit 2. That is, it prevented 'distal deepening' (Slack, 1977).Normally a polarizing region graft brings about a 50 % increase in width bystage 24 or 25, when the digits are being laid down (Figs la and 4a; Tickleet al. 1975). Irradiation inhibits widening such that, at the same time after thegraft, the limb bud is only about 15 % wider (Figs 8 a and 9 a). The change inthe 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 weretreated with 10 Gy X-irradiation at various times after the operation. Whenirradiation was delayed until 18 h after the operation, by which time somewidening had occurred, half the resulting reduplicated wings did containdigit 2. When irradiation was delayed until 28 or 42 h a typical reduplicatedwing was rarely formed but the host digit 2 was intact. It is possible that therapidly-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 manuscriptand the MRC for financial support.

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COGGLE, J. E. (1973). Biological Effects of Radiation. London and Winchester: WykehamPublications (London) Ltd.

COOKE, J. & SUMMERBELL, D. (1980). Cell cycle and experimental pattern duplication in thechick wing during embryonic development. Nature, Lond. 287, 697-701.

FALLON, J. F. & CROSBY, G. M. (1977J. Polarizing zone activity in limb buds of amniotes.In Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M.Balls), pp. 55-69. Cambridge and London: Cambridge University Press.

FRENCH, V., BRYANT, P. J. & BRYANT, S. V. (1976). Pattern regulation in epimorphic fields.Science 193, 969-981.

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ITEN, L. E. & MURPHY, D. J. (1980). Pattern regulation in the embryonic chick limb: super-numerary limb formation with anterior (Non-ZPA) limb bud tissue. Devi Biol. 75, 373-385.

KARNOVSKY, M. J. (1965). Formaldehyde-glutaraldehyde fixative of high osmolarity for usein electron microscopy. / . Cell Biol. 27, 137a.

MACCABE, J. A., CALANDRA, A. J. & PARKER, B. W. (1977). In-vitro analysis of the distri-bution and nature of a morphogenetic factor in the developing chick wing. In VertebrateLimb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls), pp. 25-39.Cambridge and London: Cambridge University Press.

MACCABE, J. A., LYLE, P. S. & LENCE, J. A. (1979). The control of polarity along the antero-posterior axis in experimental chick limbs. J. exp. Zool. 207, 113-120.

MACCABE, J. A. & PARKER, B. W. (1975). The in vitro maintenance of the apical ectodermridge of the chick embryo: an assay for polarizing activity. Devi Biol 45,349-357.

MACCABE, J. A. & PARKER, B. W. (1976). Evidence for a gradient of a morphogenetic factorin the developing chick wing. Devi Biol. 54, 297-303.

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(Received 1 July 1980, revised 15 December 1980)