J. Embryo!. exp. Morph. Vol. 65, pp. 185-197, 1981
Printed in Great Britain © Company of Biologists Limited 1981
|g5
Transfilter evidence for a zone of polarizing
activity participating in limb morphogenesis
By EERO A. KAPRIO 1
From the Department of Pathology and
Laboratory of Experimental Embryology, Department of Zoology,
University of Helsinki
SUMMARY
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 005fim, 10/*m and 80/*m,
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 10fim filters and the most with 80/tm 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.
INTRODUCTION
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.
1
Author's address: Department of Pathology, University of Helsinki, Haartmaninkatu 3,
SF-00290 Helsinki 29, Finland.
186
E. A. KAPRIO
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 &
Tahka, 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/*m, 1-0/*m and 8-0 fim pores, three types of transfilter communications can be differentiated: diffusion, cell contact and cell penetration,
respectively (Saxen, 1980).
MATERIALS AND METHODS
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 005 jim
(Np0-05), l-0/*m(NPl-0) or 8-0/mi (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
ZPA in limb morphogenesis
187
AER
Fig. 1. A schematic representation of the design of the experiment. The Zone of
Polat 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.
Table 1. Data from photographs after barrier insertion
(Values given with one standard deviation.)
Cellophane
No cases
W/L
AA
F mm
F,..mm
Np 005
20
20
0-34 ±005
71% ±7-2
104±012
0-48 ±010
0-33 ±004
70% ±8-1
0-98 ±0-24
0-50±014
Np 1 -0
16
0-33 ±005
68% ±100
0-92 ±011
0-49 ±011
Np80
17
0-28±012
74% ±11-9
O-83±O17
0-46 ±015
The ratio of the mean width (W) of the tip from the flank base line, over the length (L)
along the flank level base line of the wing bud.
The area anterioi (AA) to the barrier as a percentage of the total area of the wing bud.
The length of the barrier (F mm) as well as the portion within the wing bud (Fiumm) are
both in millimetres.
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.
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).
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
188
E. A. KAPRIO
Table 2. Wing bud growth rates and barrier movement
Cellophane
NpO-05
Npl-0
Np 80
14
19
6
10
No cases
OG (jim/h)
28 ±6
28±4
28± 12
31 ±8
17±8
14±6
16±8
12±8
Fd Om/h)
Data, with standard deviation values, calculated from comparing photographs, taken
after barrier insertion and on the following day.
The time interval between the photographs was mainly 18-22 h, but with a range of
13-32 h. The outgrowth rate (OG) of the wing tip is in /on/h.
The rate at which the barrier disappeared into the wing bud (Fd) is measured from the
shortening of the barrier portion projecting out of the wing bud.
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.
RESULTS
Although only stage-20 HH embryos were used, the L/W ratio indicates
that there were slight variations in the developmental state within the stage20 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 /.im/h,
s.D. ± 8 jLim/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).
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
189
ZPA in limb morphogenesis
Control n - 17
2
3
Normal 6
Preaxial deviation 7
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.
6
Cellophane n - 20
6
CR1
CR4
14
\ _
CR13
CR8
Fig. 3. The deletion patterns for the barrier groups. The number of cases (ri) 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.
7
EMB
65
190
E. A. KAPRIO
J-**"
4a
4b
,-
4c
\
4d
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 005 fim 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 10 /im 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.
only a humerus and a radius (Fig. 4B). Of these, two had also a rudimentary
digital cartilage suggesting proximal ray III or IV cartilage. In 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.
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
ZPA in limb morphogenesis
191
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 /mi 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 /*m
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 /mi.
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 /*m.
(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
7-2
192
E. A. KAPRIO
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 1-0 jtim pores by 3 h after the
insertion (Fig. 5 A). No cell processes were seen in the Nuclepore filter with
0-05 /.im pores even at 9 h (Fig. 5B). Light microscopic examination of the
wing buds with Nuclepore filters with 8-0 [im pores showed that whole cells
had found their way into the pores.
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).
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.
ZPA in limb morphogenesis
193
DISCUSSION
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; Hampe, 1959;
Amprino, 1975; Wolpert, 1976). The deletion level after AER removal at stage20 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; Harnpe, 1959; Barasa, 1960;
Summerbell, 19746; 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 2'PA 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
194
E. A. KAPRIO
tested only those places where they expected to find the ZPA. Summerbell
(1979) placed tantalum foil and 0-8 jim 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) (Saxen, 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 (Saxen, 1961; Crick, 1970;
Toivonen et al. 1975; Karkinen-Jaaskelainen, 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 /im and a
nominal pore size of 0-45 /im 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
ZPA in limb morphogenesis
195
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 posterioranterior 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 005fim to 8-0fim 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 (Saxen & 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 005 or 1-0ju,m. However, the pores on Nuclepore 8-0ju,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,
1-17%, for Nuclepore 1-0, 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
(1956a, 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 fim.
196
E. A. KAPRIO
The author wishes to thank Professor Lauri Saxen 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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(Received 12 January 1981, revised 30 March 1981)