Barriers were inserted into stage-20 HH chick embryo wing buds to separate the zone of polarizing activity from the anterior two-thirds of the wing bud with its overlying apical ectodermal ridge. Half of the barrier length projected out of the wing bud at insertion. Sham-operated wing buds developed only occasionally into wings with cartilage deletions. After insertion of an impermeable membrane (Cellophane), the typical wing skeleton contained only a humerus and a radius. In order to differentiate between diffusion, cell contact and cell penetration, Nuclepore filters with pore sizes of 0·05μm, 1·0 μm and 8 ·0 μm, respectively, were inserted. The typical wing skeleton after Nuclepore filter insertion was one with post-axial deletions. None, however, developed with complete distal deletions as after Cellophane. Deletions in the wing skeletons after Nuclepore insertion were the least with 1·0μm filters and the most with 8·0 μm filteis. Elevation of the apical ectodermal ridge was noted until 18 h after the insertions. In none of the groups did the ridge flatten. The results suggest that the zone of polarizing activity does have a role in normal limb morphogenesis. The mechanism by which its morphogen spreads is diffusion rather than being mediated via cell contacts.

The Saunders-Zwilling hypothesis stipulates that the proximodistal morphogenesis of the limb is controlled by a reciprocal interaction between the Apical Ectodermal Ridge (AER) and the limb-bud mesenchyme. The AER is a temporary structure present only during the morphogenetic stages of limb development. The AER does not specify limb orientation, limb type or species (Saunders, 1977). Most significantly, it does not even specify the proximodistal Jevel of limb parts (Rubin & Saunders, 1972). According to the hypothesis, the AER is elevated in form when morphogenetically active and flat when inactive.

A precisely located post-axial mesenchymal activity displays polarizing properties in limb morphogenesis. It has been named the Zone of Polarizing Activity (ZPA) (Balcuns, Gasseling & Saunders, 1970; MacCabe, Gasseling & Saunders, 1973). Unlike the AER, the ZPA is not distinguishable by morphological criteria. Its activity is tested by transplantation to a pre-axial location.

There it causes pre-axial changes, including AER elevation and pre-axial polydactyly. Like the AER, it is present in the limb bud only during the morphogenetic stages (MacCabe et al. 1973). Like the AER, it does not specify the proximodistal level of the extra pre-axial cartilages it induces (Summerbell, 1974a). Though the ZPA has dramatic effects on limb morphogenesis when transplanted, doubt has been expressed as to whether it has a role in normal limb morphogenesis. One reason has been the claim that the removal of the ZPA is compatible with normal limb morphogenesis (MacCabe et al. 1973; Fallon & Crosby, 1975; Tickle, Summerbell & Wolpert, 1975). Another reason for doubt has been the finding that the flank of the developing embryo also has ZPA-like activity (Saunders, 1977; MacCabe, Calandra & Parker, 1977). In the removal experiments, however, only the ZPA area of highest activity was removed.

Therefore it was decided to investigate the role of the ZPA in normal limb morphogenesis by placing barriers anterior to the ZPA in stage-20 HH (Hamburger & Hamilton, 1951) wing buds of the chick embryo. At this stage, the ZPA lies in the posterior third of the wing bud (MacCabe et al. 1973). Most of the AER overlies the middle third of the wing bud at this stage (Kaprio & Tähkä, 1978). It is thus the earliest stage at which a barrier can be easily interposed, in ovo, between the ZPA and the AER. Thus the flank’s ZPA-like activity also lies on the opposite side of the barrier from the AER.

Four types of barriers were used. Cellophane was the impermeable barrier. The other three were Nuclepore filters with different pore sizes. Transfilter experiments with embryonic tissues have shown that by selecting Nuclepore filters with 0·05 μm, 1·0 μm and 8·0μm pores, three types of transfilter communications can be differentiated: diffusion, cell contact and cell penetration, respectively (Saxén, 1980).

White Leghorn eggs were incubated for 3| days at 38 °C. They were fenestrated, membranes cut and the embryos kept moist with Dulbecco’s phosphate buffer. Only stage-20 HH (Hamburger & Hamilton, 1951) embryos were used. A radial incision was made (with a pair of iris scissors), to separate the posterior third from the anterior two-thirds of the right wing bud (Fig. 1). In the controls the incision was made, but nothing was inserted. Cellophane, Nuclepore filters (General Electric Co., Pleasanton, Calif.) with the pore sizes of either 0·05 μm (Np0 05), 1·0μm (NP 1·0) or 8·0μm (Np 8·0) were inserted, with approximately half of the barrier length projecting out of the wing bud. The fenestration was sealed with tape and the egg returned to the incubator. The presence of the barrier was checked 13−32 h later. The embryo was rejected if the barrier had fallen off or if the wing bud showed obvious signs of damage, e.g. opacity or haematomas. The embryos were allowed to develop to the tenth day of incubation. They were then fixed in 5 % trichloroacetic acid. Both left and right wing cartilages were stained with Alcian Green (Summerbell & Wolpert, 1973). The control group consisted of 17 cases. They had an average length/width (L/W) (see Hamburger & Hamilton, 1951) ratio of 0·31.

Fig. 1.

A schematic representation of the design of the experiment. The Zone of Polai izing Activity (ZPA) shown is as adapted from MacCabe, Gasseling & Saunders (1973). The black area is the high activity and the dotted area the lower activity; AER, the Apical Ectoderm Ridge.

Fig. 1.

A schematic representation of the design of the experiment. The Zone of Polai izing Activity (ZPA) shown is as adapted from MacCabe, Gasseling & Saunders (1973). The black area is the high activity and the dotted area the lower activity; AER, the Apical Ectoderm Ridge.

All the wing buds that had barriers inserted were photographed immediately after the insertion, and most were successfully re-photographed the next day. The size of the wing bud and the length and position of the barrier were measured from these photographs (Table 1). The photograph taken the next day was used for calculating the growth of the wing bud and the proximodistal movement of the barrier (Table 2). These data and the deletion patterns seen in the wing skeletons were analysed using an SPSS package implemented on a Burroughs 6700 computer (Nie et al. 1975).

Table 1.

Data from photographs after barrier insertion

Data from photographs after barrier insertion
Data from photographs after barrier insertion
Table 2.

Wing bud growth rates and barrier movement

Wing bud growth rates and barrier movement
Wing bud growth rates and barrier movement

From each barrier group two wing buds were fixed for transmission electron microscopy 3 h after the barrier insertion. Also two wing buds were fixed 9 h after the insertion of Np 0·05 filter. The wing buds were fixed in 2 % glutaraldehyde in 0·1 M sodium-cacodylate buffer with pH 7·3 at +4 °C overnight. They were post-fixed in 1 % osmium tetroxide for 1 h using the same buffer, pH and temperature. The wing buds were dehydrated at room temperature in serial acetones and embedded in Spurr resin. Thick and thin sections were cut with an LKB 8800 Ultramicrotome III The thick sections were stained with toluidine blue. The thin, silver sections were stained with uranyl acetate and lead citrate. The thin sections were examined in a Zeiss 9A electron microscope at 60 kV.

In each barrier group, five or six embryos were fixed in Zenker fixative at 3, 9 and 18 h after barrier insertion. After prolonged washing in running water the right and left wing buds were compared and photographed under a dissecting microscope. The extents and elevations of the AERs were especially noted.

Although only stage-20 HH embryos were used, the L/W ratio indicates that there were slight variations in the developmental state within the stage-20 HH between the groups. There was no significance between the barrier groups in the differences in the lengths of the barriers, their proportions within/without the wing bud or in the positions of the barriers (Table 1). The mean outgrowth rate of the wing buds for the whole series was 29·1 μm/h, s.D. ±8μm/h, and no group differed significantly from the others (Table 2). This implies that they were all equally viable, and that the outgrowth rate, within the time observed, does not explain the different cartilage deletion patterns observed.

In the sham-operated control group, in only a few cases, some distal digital ray cartilages were deleted (Fig. 2). In seven cases, pre-axial deviation and distal digital cartilage deformity of one of the digital rays was seen (Fig. 4A).

Fig. 2.

The deletion pattern for the control, sham-operated series. The number of the cases (n) was seventeen. The number of wing cartilages deleted in four of the wings is shown. In thirteen cases there were no deletions.

Fig. 2.

The deletion pattern for the control, sham-operated series. The number of the cases (n) was seventeen. The number of wing cartilages deleted in four of the wings is shown. In thirteen cases there were no deletions.

In the four barrier groups, not a single wing with a complete wing skeleton developed. The typical pattern for the wing skeletons after Cellophane insertion was the missing wrist and all digital cartilages (Fig. 3 A). Ten cases developed only a humerus and a radius (Fig. 4B). Of these, two had also a rudimentary digital cartilage suggesting proximal ray III or IV cartilage. Tn four cases, only a humerus, an ulna and a radius had developed, and in two of these, as above, there was a rudimentary digital cartilage.

Fig. 3.

The deletion patterns for the barrier groups. The number of cases (n) in each series is shown, after the type of barrier. The number beside each cartilage indicates the number present in each series. The number of cases with curved radius (CR) in each series is also indicated. In none of the barrier series was there a single wing with full complement of cartilages.

Fig. 3.

The deletion patterns for the barrier groups. The number of cases (n) in each series is shown, after the type of barrier. The number beside each cartilage indicates the number present in each series. The number of cases with curved radius (CR) in each series is also indicated. In none of the barrier series was there a single wing with full complement of cartilages.

Fig. 4.

Wings stained for cartilage at ten days of incubation, (a) A wing after sham operation showing pre-axial deviation of distal digit III (arrow). (b) Cellophane insertion typically causes a wing to develop that has only a humerus and a radius, (c) Nuclepore 0·05 μm pore filter insertion has caused a wing to develop with a single digital ray missing, in this case ray IV is deleted (arrow). (d) A wing after Nuclepore 1·0μm pore filter insertion. The ulna is short and blunt (arrow), the radius is curved (CR) and the distal cartilage of ray III is missing (arrowhead). The humerus is split.

Fig. 4.

Wings stained for cartilage at ten days of incubation, (a) A wing after sham operation showing pre-axial deviation of distal digit III (arrow). (b) Cellophane insertion typically causes a wing to develop that has only a humerus and a radius, (c) Nuclepore 0·05 μm pore filter insertion has caused a wing to develop with a single digital ray missing, in this case ray IV is deleted (arrow). (d) A wing after Nuclepore 1·0μm pore filter insertion. The ulna is short and blunt (arrow), the radius is curved (CR) and the distal cartilage of ray III is missing (arrowhead). The humerus is split.

Of the twenty wing skeletons that developed after Np 0·05 insertion (Fig. 3B), eight had deletions of the ulna and post-axial wrist cartilages. The digital deletion pattern of this group consisted of five cases with a single ray missing (Fig. 4C) and seven with two rays missing. In the remaining eight, some parts of one or two digital rays were missing. The digital ray IV was the one most commonly affected.

Of the sixteen cases after NP 1·0 insertion, fifteen had the ulna missing or it was very short and blunt (Fig. 3C). In thirteen of these, the radius was deformed into a curve (Fig. 4D). The number of digital rays missing was the smallest of all barrier groups. Again the digital ray IV was the one most often deleted.

The patterns of the deletions and deformities of the seventeen cases after Np 8·0 insertion were in half of the cases as after Np 1·0 and in the other half as after Np 0·05. In eight cases there was a short or missing ulna with a curved radius (Fig. 3D). The proportion of digital cartilages missing was more than in either of the other two Nuclepore groups. Also more frequently than in the other two groups, two adjacent digital rays were missing. Common to all Nuclepore groups was the fact that in no case were all three digital rays missing at the same time. The order of the five groups simplified as the percentage of missing wing cartilages was as follows: sham-operated (4% missing), Np 1·0 (26 %), Np 0·05 (31 %), Np 8·0 (49 %) and Cellophane (65 %).

. Electron micrographs revealed that the mesenchymal cell processes had penetrated through the Nuclepore filter with 1·0μm pores by 3 h after the insertion (Fig. 5 A). No cell processes were seen in the Nuclepore filter with 0·05μm pores even at 9 h (Fig. 5B). Light microscopic examination of the wing buds with Nuclepore filters with 8·0μm pores showed that whole cells had found their way into the pores.

Fig. 5.

Electron micrographs of Nuclepore filters in the wing bud with adjacent mesenchyme tissue, (a) Three hours after the insertion of Nuclepore 1·0 μm pore filter. The mesenchymal cell processes have penetrated into the pores and through the filter to the other side. (b) Nine hours after the insertion of Nuclepore 0·05 μm pore size filter. The pores are filled with extracellular material. There are no indications of cell processes penetrating into the filter. Mesenchymal cells (M), filter (f), bar = 1 μm.

Fig. 5.

Electron micrographs of Nuclepore filters in the wing bud with adjacent mesenchyme tissue, (a) Three hours after the insertion of Nuclepore 1·0 μm pore filter. The mesenchymal cell processes have penetrated into the pores and through the filter to the other side. (b) Nine hours after the insertion of Nuclepore 0·05 μm pore size filter. The pores are filled with extracellular material. There are no indications of cell processes penetrating into the filter. Mesenchymal cells (M), filter (f), bar = 1 μm.

The AER elevation and its pre-axial extent was compared with the left unoperated wing bud from the fixed embryos at 3, 9 and 18 h after operation. In no case was there a reduction in overall elevation or noticeable reduction in the pre-axial extent of the AER in any of the five groups. In the control group, in the occasional wing bud, small gaps in the continuity of the AER were seen at the site of the incision. These gaps were more frequent and wider in all the barrier groups. They were often on both sides of the barrier, and there was no preference for the posterior or anterior side of the barrier (Fig. 6).

Fig. 6.

A wing bud fixed in Zenker fluid 9 h after the insertion of a Cellophane barrier (arrowheads). The pre-axial AER (pa) remains elevated. There is a gap in the AER continuity at either side of the barrier (arrows). Bar = 200 μm.

Fig. 6.

A wing bud fixed in Zenker fluid 9 h after the insertion of a Cellophane barrier (arrowheads). The pre-axial AER (pa) remains elevated. There is a gap in the AER continuity at either side of the barrier (arrows). Bar = 200 μm.

The barrier length, proportion within and without the wing bud, its position and its proximodistal shift were compared with the subsequent deletions. These calculations were made for individual wings, for groups and for the whole series. None of the above variables explained the results obtained except for the few cases where the barrier was more than two standard deviations anteriorly. Then the radius was deleted. The deletion pattern of any single wing cartilage could not be explained by the above variables either. No barrier shifted in a proximodistal direction faster than the rate at which the wing bud grew in the same direction. Though the barriers shifted proximodistally at different mean rates, there was no significance in the differences between the groups (Table 2). Thus only the differences in the permeabilities of the barriers explain the different deletion patterns.

The sham-operated wing buds developed into wing skeletons with deletions only occasionally, thus showing that the operation as such does not cause deletion defects. It does not, however, exclude the fact that the persisting barrier as such, irrespective of its permeability, interferes at a local level with the morphogenetic behaviour of the AER and the mesenchyme, for example through changes in the local micro-environment.

In all groups the overall elevation of the AER was normal, even 18 h after the insertion. There were, however, frequently gaps on either or both sides of the barrier. The removal of a short segment of the AER is followed by segmental distal deletions of the distal limb parts (Saunders, 1948; Hampé, 1959; Amprino, 1975; Wolpert, 1976). The deletion level after AER removal at stage-20 HH is the wrist (Kaprio & Tahka, 1978; Kaprio, 1979). Thus a single digital ray deletion could be explained by a local AER damage. As the ray-I V cartilages were the most frequently deleted in all groups, this could be the result of local AER damage.

All the barriers were placed into the prospective ulna region (Stark & Searls, 1973). It has been reported that when barriers were placed into prospective humerus regions, without cutting the AER, humerus deformities were seen in the subsequent, otherwise normal skeletons (Summerbell, 1979). It seems likely that the ulnar deletions seen in this study could also be due to local interference by the barrier. Thus, it would seem reasonable to conclude that the post-axial deletions in all barrier groups may be due to local effects of the barrier irrespective of its permeability.

The total removal of the AER causes distal deletions in the subsequent limb (Saunders, 1948; Amprino & Camosso, 1955; Harnpé, 1959; Barasa, 1960; Summerbell, 1974b; Kaprio & Tahka, 1978; Kaprio, 1979). Disconnecting the ZPA from the anterior two-thirds of the wing bud with its overlying AER, as done in this study, also causes distal deletions. The deletion pattern is the same as after AER removal, when allowance is made for the local effects of the barrier.

This finding is in contradiction with the conclusions of those experiments where the removal of the ZPA was claimed compatible with the normal development of the limb (MacCabe et al. 1973; Fallon & Crosby, 1975; Tickle, Summerbell & Wolpert, 1975). However, in these experiments, only the ZPA region with highest activity was removed. It has been confirmed that the removal of only the highest ZPA activity area is compatible with normal limb morphogenesis, but the removal of all of the ZPA results in the development of a humerus and a radius only (Hinchliffe, 1981).

Fallon & Crosby (1975) doubted the role of ZPA in normal morphogenesis as they later failed to find ZPA activity in the posterior edge of the wing bud after their subtotal ZPA removal. However, their testing was limited as they tested only those places where they expected to find the ZPA. Summerbell (1979) placed tantalum foil and 0·8 μm Millipore filters into stage-16 to -18 HH and only tantalum foil into stage-20 to -22 HH chick embryo wing buds at various anterior-posterior levels. He obtained wings with segmental distal deletions, but did not discuss the possible role of AER damage. In one series only he placed the tantalum foil so that the ZPA and the AER were on the opposing sides of the barrier, according to the ZPA maps (MacCabe et al. 1973). In that series he also obtained wings with only a humerus and a radius (Summerbell, 1979, fig. 2D). MacCabe failed to find ZPA activity in the American wingless mutant as reported by Saunders (1972). Thus the evidence from the wingless mutant, from the total removal of the ZPA, and from the interposing of impermeable barriers, all being associated with distal deletion defects, strongly suggests that the ZPA has a role in normal limb morphogenesis.

The interposed membranes used in this study can be roughly classified into four categories: one preventing diffusion (Cellophane), the others allowing it but preventing actual cell contacts (Nuclepore 0·05) or cell passage (Nuclepore 1·0), and finally the type most probably allowing the passage of cells (Nuclepore 8·0) (Saxén, 1980). The fact that the main differences in their biological consequences were observed between the impermeable Cellophane and the various types of Nuclepore filters suggests that interference with a diffusible factor rather than with cell relations affected development. This suggestion is also compatible with the minor differences observed between the various Nuclepore filter groups (see below). Such diffusible morphogens have been frequently suggested in other developmental systems as well (Saxén, 1961; Crick, 1970; Toivonen et al. 1975; Karkinen-Jääskeläinen, 1978). In fact, Tickle et al. (1975) have suggested that the ZPA produces a diffusible morphogen that in the limb causes a posterior-anterior gradient to be established in a sourcesink fashion. Their suggestion is based on the patterns of polydactyly obtained if the distance between the normal and transplanted ZPA is varied. More direct evidence for a diffusible morphogen has been obtained by MacCabe & Parker (1975) using the anterior AER and the subjacent mesenchyme as an in vitro assay of ZPA activity. They found that a 4-day-old embryo wing bud had a posterior-anterior gradient of ZPA activity blocked by an impermeable barrier. However, a Millipore membrane with a thickness of 25 μm and a nominal pore size of 0·45 μm allows the passage of ZPA activity (MacCabe & Parker, 1976). With the same assay method ZPA activity was found in the supernatant from cultures of the ZPA-containing mesenchyme. This morphogen was even dialysable. However, the morphogen from a homogenate of the ZPA region is not dialysable, and other analyses indicated that it had a molecular size of over 300000 molecular weight (MacCabe & Parker, 1979; Calandra & MacCabe, 1978).

Summerbell (1979) also reached the conclusion that the ZPA exerts its effect through a diffusible morphogen from the results of his experiments with barriers in the wing buds of chick embryos. He placed them at various posterior-anterior levels as described above and obtained segmental distal deletions. He assumed that the ZPA posterior-anterior gradient as proposed by Tickle et al. (1975) had been locally disturbed. From his calculations he concluded that the morphogen is small in size and that it spreads through the intracellular compartment of the wing-bud mesenchyme via gap junctions. The present results suggest a morphogen that is also diffusible but acts in the extracellular compartment.

The fact that porous membranes allowing diffusion also caused deletion defects is partly explained by a local effect on the wing mesenchyme. However, differences in the inhibitory effects of various Nuclepore membranes with a nominal pore size of 0 · 05 μ m to 8 · 0 μ m cannot be due to local damage or irritation. There are two other variables, the pore density and pore area to be considered in comparing the filters. These variables have been shown to affect transfilter comunication in interactive systems where the suggested interaction is mediated by cell contact (Saxén & Lehtonen, 1978), by extracellular matrix (Meier & Hay, 1975) and even through diffusion (Toivonen et al. 1975). In this study, electron micrographs of the limb bud show that all the mesenchymal cells adjacent to the filter have pores next to them when the filter has a pore size of 0 · 05 or 1 · 0 μ m. However, the pores on Nuclepore 8 · 0 μ m filter are so sparse that only some mesenchymal cells adjacent to the filter are also adjacent to a pore. This is not the first observation of Nuclepore filters with large but sparse pores preventing induction more effectively (Toivonen et al. 1975). The calculated pore areas from the manufacturer’s data are: for Nuclepore 0 · 05, T17%, for Nuclepore TO, 15 · 1 % and for Nuclepore 8 · 0, 5 · 7%. Hence, it is possible that all membrane filters interposed interfered with the diffusion of the morphogen as their porosity was relatively low.

The observation in this study that the overall AER elevation was not reduced suggests that the hypothetical apical ridge maintenance factor of Zwilling (1956 a, b\ Zwilling & Hanborough, 1956) was not interfered with. This is also in accordance with the claim that the ZPA is not the maintenance factor (Saunders, 1977), and that the ZPA does not act directly or via the AER (MacCabe & Parker, 1979). The persisting normal elevation of the AER, at least until 18 h after barrier insertion, also suggests that the concept of the maintenance factor is still useful in the analysis of the pathways of limb-bud morphogenetic regulations.

The results of this study strongly suggest that the ZPA does have a role in the normal limb morphogenesis. They also suggest that the ZPA’s morphogen is diffusible in the extracellular compartment. They show that the ‘morphogen’ travels at least 10 − 20 μ m.

The author wishes to thank Professor Lauri Saxén for his helpful criticism during the course of this project. Thanks are due to Dr Jaakko Kaprio for the statistical calculations. The expert technical assistance of Mrs H. Anthonsen is gratefully acknowledged. Financial support was received from the Aaltonen Foundation, the Finnish Culture Foundation and the Foundation for Paediatric Research, Finland.

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