Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting

16 Pages·2005·2.88 MB·English

Development 101, 501-515 (1987) 501
Printed in Great Britain © The Company of Biologists Limited 1987
Posterior apical ectodermal ridge removal in the chick wing bud triggers
a series of events resulting in defective anterior pattern formation
WILLIAM L. TODT* and JOHN F. FALLONt
Department of Anatomy, The University of Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706, USA
* Present address: Department of Physiology and Biophysics, University of California, Irvine, CA 92717. USA
t Author for reprints
Summary
The ability of the anterior apical ectodermal ridge to by necrosis in the distal, anterior mesoderm at 48 h
promote outgrowth in the chick wing bud when postoperatively and subsequent anterior truncation.
disconnected from posterior apical ridge was exam- Clearly, healthy posterior limb bud mesoderm is
ined by rotating the posterior portion of the stage- needed for anterior limb bud survival and develop-
19/20 to stage-21 wing bud around its anteroposterior ment. We propose that anterior truncation is the
axis. This permitted contact between the anterior and direct result of anterior mesodermal cell death and
posterior mesoderm, without removing wing bud that this may not be related to positional specification
tissue. In a small but significant number of cases of anterior cells. In our view, cell death of anterior
(10/54), anterior structures (digit 2) formed spatially mesoderm, after posterior mesoderm removal, should
isolated from posterior structures (digits 3 and 4). not be used as evidence for a role in position specifi-
Thus, continuity with posterior ridge is not a pre- cation by the polarizing zone during the limb bud
requisite for anterior-ridge function in the wing bud. stages of development. We suggest that the posterior
Nevertheless, posterior-ridge removal does result in mesoderm that maintains the anterior mesoderm need
anterior limb truncation. To investigate events leading not be restricted to the mapped polarizing zone, but is
to anterior truncation, we examined cell death pat- more extensively distributed in the limb bud.
terns in the wing bud following posterior-ridge re-
moval. We observed an abnormal area of necrosis Key words: limb development, apical ectodermal ridge,
along the posterior border of the wing bud at 6-12 h wing bud, cell death, polarizing zone, chick, pattern
following posterior-ridge removal. This was followed formation.
Introduction & Fallon, 1982) and the specification of distal limb
elements ceases, resulting in a stage-dependent trunc-
The avian limb bud consists of a mesodermal core ation of the formed limb (Saunders, 1948; Summer-
covered with simple cuboidal epithelium on the bell, 1974; Rowe & Fallon, 1982). It is clear that
dorsal and ventral surfaces and capped apically by a normal development of the limb depends on com-
pseudostratified columnar ectodermal ridge. Inter- munication between the mesoderm and ectoderm.
actions necessary for normal limb formation occur Communication is also required along the antero-
between the mesoderm and apical epithelium. In- posterior axis of the limb. Posterior tissue develops
itially, the limb mesoderm induces formation of the normally if the anterior tissue is removed. However,
ridge (Kieny, 1960, 1968; Saunders & Reuss, 1974) anterior development is truncated if the posterior
and subsequently maintains it as well (Zwilling & tissue is removed (Warren, 1934; Hinchliffe & Gum-
Hansborough, 1956; Fallon, Boutin & Carrington, pel-Pinot, 1981). Insertion of a barrier between the
1986). Removal of the apical-ectodermal ridge results anterior and posterior tissue is also followed by
in necrosis in the subjacent mesoderm (Rowe, Cairns anterior truncation (Warren, 1934; Summerbell,
502 W. L. Todt and J. F. Fallon
1979; Kaprio, 1981). In addition to anterior trunc- experimentally isolated from posterior ridge provided
ation, cell death in the anterior mesoderm is observed that limb tissue is not removed and contact between
following these operations (see Hinchliffe & Grif- the anterior and posterior mesoderm is reestablished.
fiths, 1984). These data demonstrate that normal We also report specific cell death patterns in the
anterior development is dependent on posterior tis- mesoderm following posterior-ridge removal. These
sue. results permit insights into the ectodermal-mesoder-
A series of experiments beginning with Saunders & mal and mesodermal-mesodermal interactions oc-
Gasseling (1968) demonstrates that posterior border curring during limb development.
mesoderm can bring about polarized duplications
when a small piece is grafted into an anterior limb bud
Materials and methods
site. This has been interpreted to mean that posterior
border mesoderm establishes a gradient of a morpho- Fertile White Leghorn eggs were incubated at 37-38°C for
gen that prescribes the anteroposterior axis during 3 days, candled, windowed, sealed with Scotch magic tape
normal limb development (see Tickle, Summerbell & and reincubated until they reached the desired stage
Wolpert, 1975). Thus, in this view, normal limb (Hamburger & Hamilton, 1951). To operate on the em-
development is dependent on active production of the bryo, a small hole was made in the membranes overlying
morphogen and continuous maintenance of the gradi- the wing bud and two drops of sterile Tyrode's solution
were added. The wing bud apex was stained lightly with
ent until all limb structures are specified. The pro-
Nile blue A and a camera-lucida drawing of the wing bud
posed morphogen has not been identified. However,
and adjacent somites was made (final magnification, X290).
provocative results have been achieved with retinoic
Following each operation described below, two drops
acid (Tickle, Alberts, Wolpert & Lee, 1982; Summer- of penicillin-streptomycin (500 units ml"1; 500mcgml~';
bell, 1983; Eichele, Tickle & Alberts, 1985; Thaller &
GIBCO) were added and eggs were resealed. We observed
Eichele, 1987) in that it can mimic the action of better development of the anterior tissue in rotation exper-
grafted posterior border mesoderm and cause polar- iments if the embryos were held at room temperature for a
ized duplications. short period. Therefore, in all types of experiments, some
Summerbell (1979) and Hinchliffe and co-workers embryos were left at room temperature (25°C) for 1 to 1-5 h
(Hinchliffe & Gumpel-Pinot, 1981; Hinchliffe & Grif- after the operation while others were reincubated immedi-
ately. Only in the case of 180° posterior-bud rotation did
fiths, 1984; Wilson & Hinchliffe, 1987) argue that
room temperature affect the results (see below). Embryos
anterior defects seen after posterior-bud removal or
were checked several times in the 2 days following the
barrier insertion are due to isolation of the anterior
operation and prepared for cell death studies or allowed to
mesoderm from the polarizing influence of the pos-
develop until the skeletal patterns could be analysed.
terior mesoderm. However, their work does not rule
out the possibility that separation of the anterior Experimental manipulations (Fig. 1)
ridge from the posterior ridge is responsible for Partial bud removal
anterior truncation. If the anterior ridge became To remove part of the wing bud, a cut was made through
nonfunctional when separated from the posterior the wing bud, parallel to the proximodistal axis, with a pair
ridge, the results obtained would mimic the surgical of sharpened forceps. The location of the cut was marked
removal of the anterior ridge, i.e. cell death would on the camera-lucida drawing. A cut was then made along
occur in the distal, anterior mesoderm and truncation the base of either the anterior (Fig. 1A) or posterior
(Fig. IB) portion of the bud. The excised tissue was
of digital skeletal structures would result. As men-
removed from the egg.
tioned above, cell death and truncation are seen
following barrier insertion and posterior-bud removal
Partial ridge removal
(Hinchliffe & Gumpel-Pinot, 1981; Hinchliffe & Grif-
In order to remove a portion of the apical ectodermal ridge,
fiths, 1984). Rowe & Fallon (1981) demonstrated that
the region of ectoderm to be removed was stained lightly
posterior-ridge removal, leaving the anterior apical with Nile blue A. Two cuts were made through the
ridge and the posterior mesoderm in situ, results in ectodermal ridge using a sharpened tungsten needle. One
truncated anterior development. To understand why cut was near the boundary between digits 2 and 3 (see
such different manipulations (posterior-bud removal, below and Fig. 2). The second cut was made within 150/im
barrier insertion and posterior-ridge removal) all of the anterior (Fig. 1C) or posterior (Fig. ID) junction of
the limb bud and body wall. The locations of the cuts were
result in anterior truncation, two questions must be
marked on the camera-lucida drawing. Then the dorsal
answered. (1) Is anterior apical ridge inactivated
ectoderm proximal to the apical ridge was cut along the
when separated from posterior ridge? (2) What cell
extent of the ridge to be removed. The apical ridge was
death patterns accompany posterior-ridge removal?
teased off the apex of the bud. In the last step for ridge
We examined the ability of anterior ridge to sup- removal, the ventral ectoderm proximal to the ridge was cut
port normal anterior limb development and found and the apical ectoderm, including the apical ridge, was
that anterior ridge can support development when removed from the egg.
Posterior ridge and anterior wing bud patterning 503
Posterior-bud rotation and the posterior part of the bud was rotated 180° around
the pin. A second pin was inserted through the rotated
To rotate the posterior portion of the wing bud, a cut was
tissue and into the body wall, fixing the tissue in the rotated
made parallel to the proximodistal axis and the location was
position (Fig. IF).
marked on the camera-lucida drawing. A platinum pin
(0025mm diameter, Goodfellow Metals, Cambridge) was
Cell death studies
inserted parallel to the anteroposterior axis (Fig. IE). A cut
was made along the base of the posterior portion of the bud Regions of cell death were examined in ovo for some
embryos at each stage (19/20 through 21) and for each type
of operation. In addition, aseriesof wholemount wing buds
was prepared to compare cell death patterns following
posterior-ridge removal and posterior-bud removal at stage
20/21.
Cell death was visualized using the vital dye neutral red
(see Fallon & Saunders, 1968; Hinchliffe & Ede, 1973). To
Partial-bud
observe cell death in situ, a drop of neutral red solution
removal
(1/1000 in saline; 137mM-NaCl, 2-8mM-KCl) was dropped
in the egg and followed by a 20min incubation a t37 °C. In
ovo staining did not alter the percentage of embryos
surviving until day 10 (approx. 85 %).
Another way to observe cell death was to prepare
wholemounts. Embryos were placed in a dilute neutral red
solution (1/10000 in 0-075 M-phosphate buffer, pH7-4) and
incubated for 20-30 min at 37°C. Following several rinses in
phosphate buffer, embryos were fixed in aqueous formal-
dehyde (4 %) at 4°C, overnight. Tissue was dehydrated in
ethanol and the limbs were embedded in EMbed 812
(Electron Microscopic Sciences). Embedded tissue was
viewed en bloc, with stereopair micrographs taken for
permanent record; the blocks were then sectioned at 5 fim,
stained with azure II-methylene blue and examined histo-
logically (see Todt & Fallon, 1986). For comparing pos-
terior-bud removal with posterior-ridge removal, between
Fig. 1. Semidiagrammatic representations of stage-20/21
Partial-ride right wing buds illustrating each type of operation.
removal
Anterior is up, posterior is down and distal is to the
right.
(A) Anterior-bud removal. Anterior tissue was
removed at or slightly anterior to the boundary line
between presumptive digits 2 and 3 (see Fig. 2).
(B) Posterior-bud removal. Posterior tissue was
removed at or slightly posterior to the boundary line in
Fig. 2.
(C) Anterior apical ectodermal ridge removal. The
distal ectoderm, including the apical ridge, was removed
from near the anterior junction of the wing bud and body
wall to the level of the boundary line in Fig. 2 or slightly
anterior to it.
(D) Posterior apical ectodermal ridge removal. The
distal ectoderm, including the apical ridge, was removed
from near the posterior junction of the wing bud and
body wall to the level of the boundary line in Fig. 2 or
slightly posterior to it.
Posterior-bud
(E) First steps in posterior-bud rotation. A horizontal
rotation
cut was made as in (B) at or slightly posterior to the
boundary line in Fig. 2 and a pin (dashed line) was
inserted.
(F) Final steps in posterior-bud rotation. Posterior
tissue was cut free and rotated 180° around the first pin.
Tissue was secured in the rotated orientation with a
second pin (dashed line). Scale bar (F) represents 250jum.
504 W. L. Todt and J. F. Fallon
three and six embryos were examined at each time dis- Controls
cussed in the results. Abnormal areas of cell death were As a control for nonspecific effects of posterior-ridge
defined as those present only in the operated wing bud removal, the posterior, dorsal ectoderm was removed. In
when compared with the normally occurring zones of this way, posterior mesoderm was exposed to trauma and
necrosis in the contralateral limb bud (see Saunders, the amniotic fluid as in posterior-ridge removal. Control
Gasseling & Saunders, 1962). rotation experiments were done by rotating the posterior
tissue 360°. The tissue was pinned in its original orientation.
Finally, in the cell death studies, the unoperated left wing
Analysis of skeletal patterns bud served as a control for the appearance of abnormal cell
death in the operated right wing bud.
When embryos reached 10-12 days of incubation they were
fixed in aqueous formaldehyde (4 %), stained with Victoria
blue B, dehydrated and cleared in methyl salicylate. The Results
size and configuration of the skeletal structures in the wings
were compared with the untreated controls (left wing). We Limb bud variability
have designated structures in the operated limbs that were As one purpose of these studies was to examine the
95 % of the length of the corresponding structure in the left development of anterior tissue, specifically digit 2,
wing to be normal in size (see Summerbell & Wolpert, following operations involving posterior tissue, it was
1973). Structures that were between 85% and 95% of necessary to determine the location of the boundary
normal size were labelled as slightly reduced. Reduced between presumptive digits 2 and 3. After comparing
structures were less than 85 % of the length of the corre- a large number of normal stage-19 to -22 embryos, we
sponding structure in the left wing. Truncated skeletal noted variations in the absolute size and contour of
structures had fewer elements than normal (e.g. a digit 3 the wing bud. The profiles matching the largest
with two cartilaginous elements instead of the normal proportion of wing buds at each stage is illustrated in
three). Finally, in extreme cases it was difficult to measure Fig. 2. Only operations on stage-19/20 to -21 wing
the humerus, so the data for the humerus are qualitative buds matching these standard profiles are reported
instead of quantitative. As there was variation in the here. In this way, direct comparison of the antero-
absolute size of the stained and cleared wings, we have posterior location of different operations was possible
included the left wings in Figs 3, 6. This allows for by superimposing the camera-lucida drawings and the
quantitative comparison to the unoperated wing. appropriate standard profile.
19-20 20 20-21 21
16
20
B D
Fig. 2. Location of the boundary between presumptive digits 2 and 3. Standard profiles of right wing buds are shown
for each stage used. Anterior is up, posterior is down and distal is to the right. Stage numbers (Hamburger & Hamilton,
1951) are (A) 19/20; (B) 20; (C) 20/21; (D) 21. On the left of each figure, the approximate somite levels are indicated
(somites are labelled in A). The horizontal line in each wing bud represents the boundary between presumptive digits 2
and 3, determined from anterior-bud removal experiments. Scale bar (D, upper right) represents 250 jim. Bars
associated with the boundary lines show the limits of the regions in which operations were done, determined by aligning
the camera-lucida drawing with the standard profile (see text). For example, at stage 19/20, anterior-bud and anterior-
ridge removals were done at a level between the boundary line and a parallel line 25 ^m anterior to the boundary line.
Posterior-bud and posterior-ridge removal and rotation experiments were done between the boundary line and a parallel
line 50/mi posterior to it.
Posterior ridge and anterior wing bud patterning 505
Effects of anterior tissue removal By 36—48h following posterior-bud removal, abnor-
Anterior-bud removal mal cell death was seen in the distal, anterior meso-
derm (stippled areas in Fig. 4C,D). In addition, it was
A series of anterior-bud removals was done to deter-
obvious that the proximodistal dimension of the wing
mine the location of the boundary line between
bud was shorter than the control, left wing bud.
presumptive digits 2 and 3. The anteroposterior level
These observations are in agreement with Hinchliffe
that resulted in consistent loss of digit 2, while not
& Gumpel-Pinot (1981).
adversely affecting digit 3, is illustrated in Fig. 2
When the wing bud tissue posterior to the bound-
(about the level of the junction of somites 18 and 19).
ary line between presumptive digits 2 and 3 was
Abnormal cell death was not observed in the
removed, the presumptive digit 2 tissue was left in
posterior mesoderm following anterior-bud removal.
place, yet digit 2 did not develop in 29/31 cases. Digit
Anterior-bud removals at the levels indicated in
2 developed only when digit 3 was also present
Fig. 2 resulted in deletion of the radius (23/23 cases)
(2/31). The humerus was always reduced in size and
and also digit 2 (22/23 cases; Fig. 3A-C). The humer-
the radius was usually present (29/31 cases; see
us was reduced in size and the glenohumeral joint was
Fig. 3H). The ulna developed in one specimen with
defective (see Fig. 3B). The ulna and digits 3 and 4 digits 2 and 3, and was reduced in another specimen
were normal to reduced in size. In one case, a nodule with a cartilage nodule where digit 2 should form.
of cartilage was attached by soft tissue to the anterior Finally, in one case, there were two unidentifiable
surface of the middle third of the first phalangeal cartilaginous elements posterior to the radius.
element of digit 3. Finally, one case had a reduced
digit 2.
Posterior-ridge removal: cell death patterns
Removal of the apical ridge posterior to the boundary
line between presumptive digits 2 and 3 resulted in an
Anterior-ridge removal
abnormal region of cell death in the mesoderm. At
When only the anterior apical ridge was removed to
6-12 h following posterior-ridge removal, a large area
the level of the boundary line between presumptive
of abnormal cell death was seen in the posterior
digits 2 and 3, abnormal cell death was seen in the
mesoderm of all six wholemount specimens (stippled
anterior mesoderm within 5h (see Rowe, Cairns &
area in Fig. 4E; see also Fig. 5A). This cell death was
Fallon, 1982). In the skeletal patterns examined after
predominantly in the dorsal half of the wing bud. In
anterior-ridge removal, digit 2 failed to develop in
addition, in some wing buds there were macrophages
18/23 cases while digits 3 and 4 were not adversely
scattered throughout the rest of the wing bud (com-
affected (Fig. 3D-F). In the five cases where digit 2
pare Fig. 5A and 5B), although these were not seen
was present, it was always slightly reduced to reduced
consistently. The pattern of extensive posterior nec-
in size. The radius was absent in 18/23 cases. Two of
rosis 6-12 h after posterior-ridge removal was also
the five cases with a radius also had a slightly reduced
observed in five embryos stained in ovo.
to reduced digit 2. Thus, removal of anterior ridge
The posterior cell death pattern just described
results in anterior truncation in most cases (see also
suggests that cell death following posterior-ridge
Rowe & Fallon, 1981; Iten, 1982).
removal eliminates a significant portion of the pos-
In summary, we have located the boundary be- terior wing bud mesoderm. This is supported by the
tween presumptive digits 2 and 3. In addition, we obvious lack of development in the posterior half of
confirmed that removal of the wing bud or apical the operated wing bud compared to the contralateral
ridge anterior to the boundary line resulted in an- wing bud (see Fig. 4). Additional support comes from
terior deletions, but did not affect the formation of the observation that at 24 h following posterior-ridge
the remaining posterior skeletal structures (Hinch- removal (stage 25) the normal posterior necrotic zone
liffe & Gumpel-Pinot, 1981; Rowe & Fallon, 1981). (Saunders et al. 1962) was not seen in three right wing
buds but was evident in the unoperated left wing buds
(Fig. 4F). Cell death at 24 h following posterior-ridge
Effects of posterior tissue removal
removal was restricted to the normal anterior necrotic
Posterior-bud removal
zone (ANZ) (Fig. 4F).
Following posterior-bud removal to the level indi- At 36— 38 h following posterior-ridge removal a
cated in Fig. 2, an area of abnormal cell death was small increase in the amount of cell death along the
seen along the anterior border of the remaining wing anterior border was seen (n = 4; Fig. 4G). By 48 h, an
bud, beginning at 18 to 24 h (stippled area in Fig. 4B). area of abnormal cell death was seen extending into
506 W. L. Todt and J. F. Fallon
the distal mesoderm (n = 3; Fig. 4H) beneath the Posterior-ridge removal: skeletal patterns
remaining apical ectoderm. By 48h following either Removal of the apical ridge posterior to the boundary
posterior-ridge removal or posterior-bud removal, line between presumptive digits 2 and 3 resulted in
the shape and cell death pattern of the resulting wing truncated anterior development (Fig. 3J-L). Even
buds were very similar (compare Fig. 4D and 4H). though some of the metacarpal for digit 2 was often
3A B
H
Posterior ridge and anterior wing bud patterning 507
present, digit 2 failed to form in all 29 cases. The Dorsal ectoderm removal
radius was always present and development of the As a control for posterior-ridge removals, the pos-
ulna was variable (Fig. 3K). We stress that the ridge terior dorsal ectoderm was removed, leaving the
normally associated with digit 2 and all of the wing apical ridge intact. Massive necrosis was never ob-
bud mesoderm, including polarizing zone, were left in served in the posterior mesoderm. Neutral red stain-
situ in these experiments, yet digit 2 did not form. ing was seen in the ectoderm around the edge of the
wound at 6—12h. In addition, occasional, superficial
In another nineteen embryos, we observed prox-
macrophages were observed in the mesoderm. The
imodistal outgrowth along the posterior portion of
wing buds maintained normal dimensions and devel-
the wing bud at 24-48 h following posterior-ridge
oped normal skeletal patterns in all ten cases (see also
removal. We regard this outgrowth as evidence of
Saunders, 1948; Rowe et al. 1982; Martin & Lewis,
posterior-ridge activity and we infer that the posterior
1986). This control makes unlikely the possibility that
ridge had not been completely removed. In fact,
the amniotic fluid or surgical trauma caused the
digits 3 and/or 4 were present, albeit reduced or
mesodermal cell death and truncation observed fol-
truncated, in 11/19 cases. These cases are not in-
lowing posterior-ridge removal.
cluded in our analysis except to note that, as in
posterior-bud removals, digit 2 only formed in cases Rotation of the posterior wing bud tissue
where posterior digits also developed. The rotation experiments described in the Materials
Therefore, we have confirmed that removal of the and methods section were designed to investigate the
ability of the anterior ridge to function when separ-
posterior bud (Hinchliffe & Gumpel-Pinot, 1981) or
ated from the posterior ridge. When the posterior
only the posterior ridge in the wing bud (Rowe &
wing bud tissue was rotated 180° around its antero-
Fallon, 1981) results in truncated anterior develop-
posterior axis (Fig. IF), continuity was maintained
ment; digit 2 does not form. These results are in
between the anterior and posterior mesoderm. In
contrast to the development of digits 3 and 4 from the
addition, no tissue was removed from the wing bud.
posterior tissue in the absence of anterior tissue.
However, the anterior apical ectodermal ridge was
spatially separated from the posterior-apical ridge. In
Fig. 3. Skeletal patterns resulting from preliminary this way, it is possible to test directly the ability of
experiments at stage 20/21. The first column (A,D,G,J) anterior ridge to function when it is not in contact
illustrates the type of operation. The second column
with the posterior apical ridge while permitting con-
(B,E,H,K) illustrates the resulting skeletal pattern of the
tact between anterior and posterior wing bud meso-
operated right wing. The third column (C,F,I,L)
derm.
illustrates the left wing from each embryo for comparison
purposes. The normal wing skeletal elements are labelled
Skeletal patterns following posterior-bud rotation
in (C) and include the humerus (h), radius (r), ulna (u),
digit 2, digit 3 and digit 4. In addition, pectoral girdle Control 360° rotations
elements are shown: coracoid (c) and scapula (s). When tissue posterior to the boundary line between
(A,B) Anterior-bud removal. In the specimen presumptive digits 2 and 3 was rotated 360° and
illustrated here, the ulna and digits 3 and 4 were slightly
pinned in its original orientation, there was no loss of
reduced. Digit 2 and the radius were missing. The
skeletal structures. Five of seven wings had normal
humerus was reduced and the glenohumeral joint
skeletal patterns; digits 3 and 4 were slightly reduced
defective. The scapula and coracoid failed to separate.
in size (Fig. 6A,B), while digit 2 was normal to
(C) Left wing for B with skeletal elements labelled (see
reduced in size. Two of seven cases had a duplication
above).
(D,E) Anterior-ridge removal. Digit 2 was deleted in of the distal portion of digit 3. In addition, immediate
18/23 cases. The radius was reduced or absent. reincubation or holding at room temperature for
(F) Left wing for E. Note that both left and right wing about lh did not alter the skeletal pattern, nor did
buds were smaller than the others pictured. leaving the pins in or removing them after healing of
(G,H) Posterior-bud removal. The humerus and radius the graft. Thus, the manipulations involved in ro-
were reduced in size. The ulna and digits 3 and 4 were tation of the posterior tissue did not cause deletion of
eliminated. In addition, digit 2 failed to develop even any skeletal structures.
though presumptive digit 2 tissue was not removed.
(I) Left wing for H.
Posterior rotation and immediate reincubation
(J,K) Posterior-ridge removal. The humerus and radius
When the posterior tissue was rotated 180° (Fig. IF)
developed normally. The radius was normal to slightly
and the eggs were immediately reincubated at 37°C,
reduced in size. Digits 3 and 4 as well as digit 2 did not
the most common result (16/22 cases) was a wing with
develop. Development of the ulna was variable.
(L) Left wing for K. Scale bar represents 2 mm for a humerus and radius growing from the anterior
illustration of skeletal patterns. tissue. The ulna and digits 3 and 4 grew posteriorly
508 W. L. Todt and J. F. Fallon
Posterior-bud removal Posterior-ridge removal
10 h
24 h
36h
D
Fig. 4. Camera-lucida drawings of wing buds at the indicated times following posterior-bud removal (A-D) and
posterior-ridge removal (E-H). The outer solid line of each figure is a reversed outline of the control left wing bud. The
inner solid line is the profile of the operated right wing bud superimposed on the reversed left wing bud profile. The
solid black areas are regions of cell death common to both wing buds (ANZ, anterior necrotic zone, Saunders,
Gasseling & Saunders, 1962). The stippled areas are regions of abnormal, excessive cell death seen in the right wing bud
resulting from the operation.
(A-D) Posterior-bud removal. (A) At 10 h following posterior-bud removal, no cell death was observed in the right
wing bud. (B) By 24 h, there was excessive cell death along the anterior border of the bud. In the left wing bud, the
normal posterior border necrosis was seen (dashed line). (C,D) At 36 and 48h following posterior-bud removal, massive
cell death remained visible in the distal mesoderm. Also note that proximodistal outgrowth was reduced.
(E-H) Posterior-ridge removal. (E) At lOh following posterior-ridge removal, massive cell death was seen in
posterior mesoderm (compare with Fig. 5A,B). (F) By 24 h, posterior cell death was no longer visible, even though
normally occurring necrosis was seen along the posterior border of the left wing bud (dashed line). The ANZ was
slightly enlarged. (G) At 36h after posterior-ridge removal, abnormal cell death was seen distal to the ANZ. (H) At
48 h after posterior-ridge removal the entire distal tip of the wing bud was necrotic (compare with Fig. 5C). Note the
similarity in size of the bud and pattern of necrosis in C, D, and H. Scale bar (bottom) represents 1 mm.
Posterior ridge and anterior wing bud patterning 509
from the region of the elbow (similar to Fig. 6C). The Table 1. Development* of presumptive digit 2
distal portion of the humerus could also be identified
Stages
'proximal' to the ulna in some cases. The rotated
Experiment (criteria) 19/20 20 20/21 21
(posterior) elements were often reduced in size.
Three of 22 cases showed very reduced development Posterior tissue removalt
of the posterior tissue, and one case showed develop- (digit 2; no digit 3)
Posterior-bud 0%(6) 0% (6) 0% (13) 0% (6)
ment of digit 3 in both the anterior and posterior
removal
tissue (similar to Fig. 6G). In two of 22 cases, a
Posterior-ridge 0%(8) 0%(6) 0% (12) 0% (3)
reduced digit 2 was seen at the distal tip of the radius removal
(similar to Fig. 6E). This is in dramatic contrast to the
complete failure of digit 2 to develop from the Posterior-bud rotation t
corresponding tissue following posterior-bud or pos- (anterior digit 2; no anterior digit 3)
terior-ridge removal. Immediate 0 % (5) 0 % (9) 33 % (6) 0 % (2)
reincubation
The healing time of these grafts, as evidenced by
Brief 25°C 20% (5) 0% (6) 27% (11) 40%-(10)
reestablishment of the venous circulation and drain-
incubation
ing of the pooled blood, was variable, being around
* Data are expressed as percent (number of observations) of
24 to 30 h in cases where it was documented. In an
wings with digit 2 developing isolated from other digits.
attempt to facilitate healing, some embryos were left
t Difference in development of isolated digit 2 following
at room temperature (25°C) for about lh following posterior tissue removal (0/60 cases) and postenor-bud rotation
the operation, prior to reincubation at 37°C. These (10/54 cases) is statistically significant (*2; 0-001<P< 0-005).
results are discussed below.
Cell death patterns following posterior-bud rotation
Posterior rotation and brief room temperature
Cell death patterns in the anterior mesoderm follow-
incubation
ing posterior-bud rotation were variable, ranging
Maintaining the embryos at room temperature appar-
from the normal ANZ to massive cell death. We infer
ently facilitated attachment and subsequent healing
that the variability in anterior cell death correlates
of the rotated tissue. First, it was often possible to
with the variable degree of anterior development
remove both pins without displacing the graft, prior
following posterior-bud rotation. Cell death was not
to reincubation at 37°C (see MacCabe & Parker,
observed in the posterior mesoderm following ro-
1979). Second, the removal of blood pooled in the
tation.
graft was completed more rapidly, sometimes as early
as 10h following reincubation.
Discussion
The range of skeletal patterns did not differ com-
pared with the immediate reincubation group. How-
The results described in this report answer the two
ever, there was an increased incidence of digit 2
questions posed in the Introduction. First, we demon-
developing isolated from the other digits (8/33 cases;
strated that anterior apical ectodermal ridge can
Fig. 6E). Thus, it is clear that digit 2 can develop
function without being connected to posterior ridge.
when posterior tissue has been rotated and no tissue
Second, we described a sequence of cell death pat-
has been removed. There were also 4/33 cases where
terns following posterior-ridge removal. Initially,
digit 3 developed from both the anterior and pos-
abnormal necrosis was observed in the posterior
terior portions of the bud (Fig. 6G).
mesoderm as a result of posterior-ridge removal.
The development of anterior tissue (presumptive Posterior mesodermal necrosis was followed by cell
digit 2) following posterior tissue removal versus death in the distal, anterior mesoderm and sub-
posterior bud rotation is contrasted in Table 1. An sequently, by anterior truncation. Clearly, anterior
isolated digit 2 never developed (0/60 cases) in either structures fail to develop because cell death elimin-
the absence of the posterior portion of the wing bud, ates the presumptive tissue for these structures.
or in the absence of only the posterior apical ectoder- A question that remains is: how does posterior
mal ridge. In contrast, digit 2 development was mesoderm contribute to survival of the anterior
observed in 10/54 cases following posterior-bud ro- mesoderm? The possibility that the polarizing zone
tation. This represents a statistically significant differ- prevents anterior cell death has been raised (Hinch-
ence {x2, 0-001</>< 0-005). Since anterior ridge liffe & Gumpel-Pinot, 1981). However, we suggest
must be active if digit 2 is to develop, these results that maintenance (survival) of the anterior mesoderm
demonstrate that anterior apical ridge can function may be separate from the polarizing property of
when experimentally isolated from posterior apical posterior mesoderm and develop our reasons for this
ridge. suggestion below.
510 W. L. Todt and J. F. Fallon
The role of the polarizing zone has been proposed giving the polarizing zone a
Polarizing zone mesoderm is defined by its ability to central role in establishment of the anteroposterior
induce the formation of polarized supernumerary pattern of the limb (see Introduction). In addition to
structures from anterior wing bud tissue (see Mac- the patterning function, a second function has been
Cabe, Gasseling & Saunders, 1973; Honig & Sum- attributed to the polarizing region, namely, that of
merbell, 1985; Hinchliffe & Sansom, 1985). A model maintaining anterior mesodermal viability (Hinchliffe
B