A quantitative analysis of the effect of excision of the AER from the chick limb-bud
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J. Embryo!, exp. Morph. Vol. 32, 3, pp. 651-660, 1974 651
Printed in Great Britain
A quantitative analysis of the effect of excision
of the AER from the chick limb-bud
By DENNIS SUMMERBELL 1
From the Department of Biology as Applied to Medicine
The Middlesex Hospital Medical School
SUMMARY
The effect of removal of the apical ectodermal ridge from the early chick limb-bud is
re-examined using a new quantitative method of analysis of results. The concept of the
proximo-distal sequence of laying down of parts is confirmed and evidence is presented
that this proceeds as a continuous process, there being a gradual change in the level specified
from one cell to another at a more distal level. The results are then interpreted in terms of
the 'progress zone' model to show that they are both consistent with the model and that
they provide an assay for one of its parameters, the rate of change of positional value with
time at the tip.
INTRODUCTION
The early development of the chick limb-bud involves three main factors:
growth, pattern formation and differentiation. To understand morphogenesis
one needs to know how the form of the early limb is obtained, then how
positional information is specified in the rudiment; so that finally, cells knowing
their place in the limb-bud may interpret this so as to differentiate accordingly
giving the normal limb morphology.
The central importance of the AER (apical ectodermal ridge) in this process
has been recognized since the classical experiment of Saunders in 1948. If the
AER is excised from an early wing-bud then no distal parts are formed. The
later the stage of the embryo at the time of operating, the more distal the plane
of trunction in the developed limb (see also Amprino & Camosso, 1955, 1956).
This experiment, together with results from carbon marking (Saunders, 1948;
Hampe, 1959) experiments, led to the concept of the proximo-distal sequence
of laying down of parts. In a paper of crucial importance, Rubin & Saunders
(1972) demonstrated that during development there is no change in the influence
of the AER on the mesenchyme and that the effect of an early ridge is in-
distinguishable from that of a later ridge. This clearly precludes the possibility
of the specification of more and more distal parts by a changing signal from
the AER.
1
Author's address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London, W1P 6DB, U.K.652 D. SUMMERBELL
More recently a model has been proposed in which the specification of
positional information is dependent on outgrowth (Summerbell, Lewis &
Wolpert, 1973). It was suggested that the presence of an AER confers special
properties on a narrow strip of mesenchyme cells near to the distal tip - the
progress zone. Cells in the progress zone, with time, experience an autonomous
change in their positional value towards a more distal level. Due to cell
division some cells are pushed further away from the AER and as they leave
the progress zone their positional value is fixed at the level which individual
cells had achieved during their stay in the progress zone. This results in a
gradient of positional value along the proximo-distal axis, which is a function
of the time a given cell lineage spent in the progress zone. In the terms of the
model, apical ridge removal halts the change of positional value in the apical
mesenchyme. The cells at the tip lose their lability prematurely and will later
differentiate at a level appropriate to their current positional value.
In order that this hypothesis may be pursued further it has been necessary
to develop a quantitative assessment of differences between perturbed and
unperturbed limbs. Recently, Summerbell & Wolpert (1973) have shown that
there is a very little variation between the lengths of skeletal elements in left
and right normal wings. It is, therefore, possible to use an observed discrepancy
in length between operated (right) and control (left) limbs as an assay of the
effect of perturbation. This method of analysis is used in this paper to examine
the effect of removal of the apical ridge both on the growth of the limbs and
on the characteristics of the proposed gradient of positional value along the
proximo-distal axis.
METHODS
Fertilized White Leghorn eggs were incubated at 38 °C and windowed on
the 3rd-7th days of development. Embryos were prepared from stages 18 to
28 (Hamburger & Hamilton, 1951).
The apical ectodermal ridge was teased away from the mesenchyme along
the entire length of the curved distal tip of the right limb-bud using fine
tungsten needles (see Fig. 1). The embryos were then returned to the incubator.
In most cases, the eggs were removed from the incubator on the tenth day
of incubation, the embryos sacrificed and the wings from operated (right) and
control (left) sides fixed in 5 % TCA, stained in Q-l % Alcian green 8GX in
70 % alcohol with 1 % hydrochloric acid, dehydrated and cleared in methyl
salicylate. Operated and control limbs were examined and photographed using
a Zeiss Stereo IV dissection microscope and the lengths of humerus, ulna,
radius and the elements of digit III measured where present (as described in
Summerbell & Wolpert, 1973). A series of operations from stage 18 were
examined repeatedly during the first 48 h after development. The lengths of
the operated and control wing-bud were measured using the perpendicular
distance between a line at the base of the limb-bud and the distal tip. ByExcision of the AER from chick limb-bud 653
3
3
* 3
Stage 21 limb Excise AER
B
c
Fig. 1. A, Diagram of the operation at about stage 21, viewed from the dorsal
aspect of the wing. B, Longitudinal section of the wing 8 h after removal of the
AER. Magnification x 400. C, Detail of longitudinal section of the wing sacrificed
immediately after operating. The apical ridge has been cut together with the bulk
of the wing. In this section, no mesenchyme cells still adhere to the AER but in
other sections occasional single cells could be found. Magnification x 600.
42 EMB 32654 D. SUMMERBELL
Table 1. The number of cases truncated at a given level
for each stage at the time of operating
Level of truncation
A
Proximal ^
Ulna/ Meta- phalanx Distal
phalanx
Stage Shoulder Humerus radius Wrist carpal digit III digit III
18 2 3 5
19 2 9 — — — —
20 — — 17 — — — —
21 — — 1 9 — — —
22 — — 1 8 — — —
23 — — — 7 — — —
24 — — — 6 2 — —
25 — — — 1 9 — —
26 — — — 2 10 — —
27 — — — — — 9 —
28 — — — — — 2 5
stage 26 this estimate of growth was discontinued as the developing elbow
joint was beginning to change the original simple outgrowth into a more
complex shape with a bend.
A further series of operations from several different stages were sacrificed
at varying periods up to 24 h after operating. They were fixed in Karnowski's
fixative, dehydrated and mounted in Araldite. 1 /«n thick sections were cut in
a plane containing the proximo-distal and dorso-ventral axes of the limb, and
stained in toluidine blue. These were used to ascertain how cleanly the operation
had been carried out.
RESULTS
There was an obvious relationship between the stage at excision and the
parts of the skeleton present in the whole mounts (Table 1). These varied
from cases (see Fig. 2) in which there were no cartilage elements (stage 18) to
those in which only the final element of digit III was affected (stage 28). It is
important to note that in the majority of cases the terminal bone present was
significantly shorter than in the contralateral control element. These elements
Fig. 2. Photomicrographs of whole limbs fixed on the tenth day of incubation,
stained with alcian green 8GX and cleared in methyl salicylate. A, AER removed
at stage 18, level of truncation: mid-humerus. B, AER removed at stage 19, level
of truncation: elbow joint. C, AER removed at stage 20, level of truncation: mid
ulna/radius. D, AER removed at stage 20, level of truncation: wrist. E, AER
removed at stage 21, level of truncation: wrist parts present. F, AER removed at
stage 25, level of truncation: metacarpals. G, AER removed at stage 27, level of
truncation: proximal phalanx of digit III. H, Normal limb.Excision of the AER from chick limb-bud 655
42-2656 D. SUMMERBELL
- A
L
0
0 10 20 30 40 50 60 70 80 90 100
" o of normal length of ulna or radius
8 ~ B
6
4
2
0 L
0 10 20 30 40 50 60 70 80 90 100
% of normal length of element of digit III
Fig. 3. Frequency distributions showing the number of embryos in which there
was a given percentage difference between the lengths of operated (right) and
control (left) skeletal elements. In normal embryos this difference would be less
than 5% in 99% of all cases. A, Ulna and radius for all limbs in which the ulna
and/or radius was the terminal skeletal element or in which there was no ulna and
radius but a normal length humerus. B, Metacarpals and carpals of digit III for
all limbs in which one of these was the terminal skeletal element. Both histograms
demonstrate that there is an apparently equal probability of obtaining terminal
elements truncated at any particular level.
did not normally have an epiphysis at the distal end but were obviously truncated
at some point along the diaphysis. Proximal ends of such elements were always
normal. No element was ever significantly longer than in the control wing.
In the histograms (Fig. 3) illustrating the frequency of given percentage
differences between operated and control sides for various chosen elements,
there were interesting frequency distributions. Non-terminal elements were
invariably of normal length and morphology. Terminal elements did not
exhibit any regulative tendency, i.e. tendency to be of normal length. The
gross level of truncation became steadily more distal the later the time of
operation.
Examinations of sections of limbs showed that the experimental technique
was not perfect. Removal of the AER was almost invariably complete, butExcision of the AER from chick limb-bud 657
2800 -
2400
Z 2000
o
c.
J 1600
O
V)
•i 1200
5
'- S00
Q 400
16 24 32 40 48
Time after operating (h)
Fig. 4. The rate of outgrowth of operated and control side limbs.
# , Control; O> operated.
occasionally tiny fragments were left. It was difficult to avoid removing frag-
ments of mesenchyme with the AER. This contamination could only be
ascertained by examining sections of the apical ridge. Sections of the rest
of the limb invariably showed neat profiles at the cut surface of the mesenchyme
which appeared undisturbed (see Fig. 1B and C).
The effect of excision was also examined by comparing outgrowth of left
(control) and right (operated) limbs. The results are shown in Fig. 4. The
length of the control limb increases linearly with age. In operated limbs
immediately following excision, the rate of outgrowth is reduced so as to
assume a much less steep slope than that for normal limbs.
DISCUSSION
The findings of this experiment confirm, in principle, those of Saunders
(1948). There is a clear relationship between the stage at operating and the
parts of the skeleton formed in the developed limb (see Table 1). If the results
are described in terms of the proximo-distal sequence of laying down of parts
then the following series may be suggested: pre-stage 19 gives humerus (Fig. 2 A
and B), stages 19-20 give ulna and radius (Fig. 2C and D), 21-24 give wrist658 D. SUMMERBELL (Fig. 2E), 25-26 give the metacarpal of digit III (Fig. 2F), 27 the proximal phalanx (Fig. 2G), and 28 the distal (Fig. 2H). It is worth emphasizing that the AER appears to be of importance to the development of distal parts long after the onset of necrosis in the interdigital zone. One surprising feature of this time course is the relatively long time spent laying down a relatively small part of the wing. While the ulna and radius (length at 10 days 4-5 mm) are laid down between stages 19 and 21 (say about 12 h), the specification of the wrist (length at 10 days 0-4 mm) takes from stage 21 to stage 24 (say about 24 h) (see Table 1). This paradox may be explained by two inter-related arguments. The wrist is a region of great complexity with many elements crammed into a short space; one should therefore expect it to correspond to a large span of positional values. If positional value changes with time at a steady rate, it should take a long while to specify the wrist, giving a long rudiment. This then requires the second part of the argument to explain how the wrist comes to be proportionally shorter than the rest of the wing after differentiation. Preliminary work in our laboratory by Julian Lewis suggests that division, after exit from the progress zone, ceases sooner than elsewhere and that the cells in the presumptive wrist region are very tightly packed. This means that although many elements are initially specified, the space they occupy eventually is very small. The quantitative analysis of the size and form of the terminal element in the truncated limb provides us with further information about the way in which the proximo-distal axis is specified. We can certainly discard the pos- sibility that the limb is laid down as a series of sub-fields equivalent to say upper arm, forearm, wrist and hand which subsequently control their own development autonomously. If this were the case, then one would expect to find that terminal elements were usually short but of normal morphology. The results clearly demonstrate that this is not the case. In the majority of embryos when the terminal element was too short it was also of abnormal morphology, lacking the distal epiphysis. One may also rule out the possibility that the loss of distal parts is due to a competition for available cells in which the proximal epiphysis always wins. The removal of whole slices of mesenchyme from a proximal level often results in the loss of only the proximal epiphysis of the bone (Summerbell, in preparation). Thus the evidence supports the notion that positional value is not specified in a series of steps equivalent to the major levels (upper arm, forearm, wrist and hand) but in a more continuous gradient along the length of the whole limb. The data on the differences between the lengths of skeletal elements in operated and control limbs (see below) confirm this impression, at least as regards the change in positional value within a single element. There is normally very little variation (± 5 %) between the lengths of skeletal elements in left and right limbs from the same embryo (Summerbell & Wolpert, 1973). The frequency distributions in Fig. 2 show that following excision of the AER most terminal elements are significantly shorter
Excision of the AER from chick limb-bud 659 than normal. In fact the distributions approximate well to rectangular, i.e. the form expected if all levels of truncation within the segment are equally probable. The length of a truncated skeletal element is directly proportional to the time between the start of specification of that element and removal of the AER. It is important that the probability of obtaining a normal length element is not high (except at the level of the wrist). Thus it seems possible that there is a continuous gradual change in the level specified from one cell to another cell at a more distal level. In this context it is interesting that the accuracy of specification of a skeletal element is equivalent to plus or minus one cell in twenty and that the length of an element at the time of initial specification is equivalent to about 20 cells (Summerbell & Wolpert, 1973). The precision achieved in morphogenesis could thus correspond to a step size of one cell length, or virtually a continuous gradient. Following removal of the AER, the limb at 10 days is short, it is also of abnormal morphology (Fig. 2). Its length is not reduced by proportionately reducing the size of all the skeletal elements, but by deleting the appropriate distal elements. Non-terminal elements were invariably of normal length compared to the control side. Even terminal elements of normal morphology (i.e. not truncated at some point along the diaphysis) were usually the correct length or only slightly shorter than the equivalent on the contralateral side. How then is the rate of outgrowth slowed (see Fig. 4) and how do parts of distal positional value fail to be specified? Janners & Searles (1971) have com- mented on the extensive cell death present in limbs from which the AER has been removed. This takes the form of a wave of cell death spreading proximally from the distal tip shortly after excision. Although this clearly must be taken into consideration when considering the reduction in outgrowth the results in this paper suggest (as do Janners & Searles themselves) that it cannot be the sole cause. Otherwise one would expect that both terminal elements of normal morphology and even more proximal elements would be shorter than usual. Indeed because of these results one must suppose the existence of some regulatory mechanism to compensate for the loss of presumptive cartilage cells. Such a mechanism could be density dependent cell division as proposed by Summerbell & Wolpert (1972). Loss of cells and the consequent reduction in cell density would be compensated for by a subsequent rise in the division rate. What of the loss of distal parts ? A plausible explanation of the results in this paper may be made if they are interpreted in terms of the progress zone model (Summerbell et al. 1973). When the apical ectodermal ridge is removed the 'progress zone' disappears. Cells at the tip stop autonomously changing their positional value and become fixed. Excision of the AER becomes then an assay for the positional value at the time of operating. The most distal level present in the stump is the level achieved in the progress zone up until the time of operating. It is important to realize that this simple relationship
660 D. SUMMERBELL
is modified by an unspecified parameter. The model does not state how quickly
the influence f the AER dissipates. At one extreme the progress zone may
disappear immediately. Alternatively, there may be a slow decline so that cells
still leave the progress zone in an orderly fashion, but rather more rapidly
than normal as its proximal boundary approaches the tip. The data suggest
that the latter is more nearly the case. If the progress zone were abolished
instantaneously a large number of cells, the entire contents of the zone, would
be specified at the level they possessed at the time of operating. If this positional
value were that of the distal end of a skeletal element then that terminal
element would have far too many cells. In fact, no embryo ever possessed on
the operated side an element which was significantly longer than on the control
side, which argues in favour of the less catastrophic change.
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