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DEVELOPMENTAL BIOLOGY 50, 16-25 (1976)
Inductive
Supernumerary
JOHN
Department
Activity and Enduring Cellular Constitution
of a
Apical Ectodermal
Ridge Grafted to the Limb Bud of
the Chick Embryo’
W. SAUNDERS, JR.,~ MARY T. GASSELING,
GfBiological
Sciences,
State University
of New York
Accepted
November
AND JANICE
at Albany,
Albany,
E. ERRICK~
New
York
12222
6, 1975
Apical
ectodermal
ridges (AERs)
isolated
from 3- to 4-day chick and quail embryos
were
prepared
by means of trypsinization
and microdissection
and then were grafted
to the dorsal or
ventral
side of a host chick wing bud. They induced
supernumerary
limb outgrowths
from the
host bud showing,
respectively,
a bidorsal
or biventral
organization,
as determined
by the
patterns
of feather
germs. The grafted ridge cells persisted,
as revealed
by histological
sections
of supernumerary
chick limb parts growing
under the influence
of quail AERs, whose cells are
readily
distinguished
after application
of the Feulgen
reagent.
These results
show that the AER induces
limb outgrowth
regardless
of whether
it is
associated
with dorsal
or ventral
limb
ectoderm
and that its continued
existence
is not
dependent
on contributions
of ectodermal
cells from the opposed ectodermal
faces of the limb
bud. The AER is pictured
as maintaining
the subjacent
mesoderm
in a condition
of developmental
plasticity
without
specifying
its differentiation
with respect to the proximodistal
axis.
It remains
uncertain
whether
the positional
values of cells that develop
under the influence
of
the AER arise within
these cells themselves
or appear in response
to influences
from proximal
sources.
INTRODUCTION
only a single AER is present, and this is
essential for limb-bud outgrowth (Saunders, 1948; Zwilling, 1949). In the mutant
eudiplopodia, two AERs arise, one proximal to the other, and in these two limb tips
are formed (Goetinck, 1964; Fraser and
Abbott, 1971a,b).
The experiments reported here were initiated some years ago to test whether a
chick limb bud developing in. situ would
respond to the graft of a supernumerary
AER dissected free from its adjacent dorsal
and ventral ectoderm. A positive result
would reinforce the view, then disputed by
Amprino (1965; see also Amprino and Ambrosi, 19731,that the AER induces the outgrowth of the limb-bud mesoderm.
Positive results were obtained as expected and as hereinafter documented.
They were noted briefly by Saunders and
Gasseling in a review paper published in
1968. These results, however, also contribute insights into other questions that were
not involved in the initial formulation of
Some years ago, Zwilling (1956b) reported that a single mesodermal core of a
3-day embryonic chick limb bud, encased
in two ectodermal hulls from donor limb
buds, forms two limb outgrowths instead
of one when grafted to a host embryo. In
view of earlier experiments from the laboratories of Saunders and of Zwilling (reviewed most recently by Saunders, 1972),
this result was attributed to the fact that
the double ectodermal component supplied
two apical ectodermal ridges (AERs) to the
recombinant. In genetically normal fowl
’ This investigation
was supported
by Grants
GM
09996, 8Tl/HD-27,
and R01 HD 07390-01A
from the
National
Institutes
of Health
and by Grant
G-14439
from the National
Science Foundation.
2 Direct requests
for reprints
to Professor
John W.
Saunders,
Jr., Department
of Biological
Sciences,
State University
of New York
at Albany,
Albany,
N.Y.,
12222.
3 Present
address:
Department
of Molecular,
Cellular and Developmental
Biology,
University
of Colorado, Boulder,
Cola. 80302.
16
Copyright
All rights
0 1976 by Academic
of reproduction
in any
Press, Inc.
form
reserved.
SAUNDERS,
GASSELINC
AND ERRICK
the experiment. One of these questions is
whether the AER is composed of a transient population of cells derived from the
distalward shifting of the ectodermal faces
of the limb bud. This view has been argued
by Amprino and his associates (1973). Another question has to do with the origin of
the dorsoventral organization of the limb
bud, a matter already treated in the context of other experiments by MacCabe,
Saunders, and Pickett (19731, Pautou and
Kieny (19731, MacCabe, Errick, and Saunders (19741, and Errick and Saunders
(1974).
In this paper we show: (1) that a supernumerary AER provided microsurgically
to a limb bud in ouo will induce supernumerary distal limb parts; (2) that the AER
is not dependent for its continued existence on the distalward sliding of the dorsal
and ventral ectodermal faces of the limbbud; and (3) that the kind of integumentary derivatives found on limb parts resulting from the presence of an additional
AER is determined by the side of the limb
on which the graft is placed. In connection
with these results we also raise the problem of the source of regional differentiation of the proximodistal axis as made possible by the presence of the AER.
Supernumerary
AER
17
30 min at room temperature. They were
then removed to a mixture of 20% horse
serum in Tyrode’s (HS-T) held at 3-4°C.
The ectodermal hull was stripped in one
piece from each mesodermal core and the
AER was isolated by trimming away the
dorsal and ventral ectoderm by means of
fine steel needles. The AER was then
transferred to the host embryo, sometimes
becoming lost or torn in the process.
Our current practice for preparing isolated AERs diminishes the chances for
damaging or losing them during transfer
to the host embryo. Using limb buds
freshly isolated in Ringer’s solution, the
ridge, together with a minimum of adjacent ectoderm and subjacent mesoderm, is
mechanically trimmed from the bud by
means of a microscalpel. It is then rinsed
in CMF and transferred to 2% trypsin in
CMF at 6-10°C for 10 min. Next it is rinsed
in HS-T (1:2) to stop enzyme action and
transferred to the host embryo. The mesodermal cells are then gently scraped away
from the ridge, which is thereupon ready
for grafting. This is the procedure that was
followed in preparing the quail AERs used
in the present experiments.
The use of AERs from chick leg buds or
limb buds of quail embryos provides a good
control for the possibility that induced suMATERIALS
AND
METHODS
pernumerary limbs might arise from doThe experiments were carried out on nor mesodermal cells contaminating the
White
Leghorn
embryos from local grafted ridge (cf. Rubin and Saunders,
sources, initially (1964) in the vicinity of 1972). No leg-specific structures appeared
Milwaukee, Wisconsin, and later (1973- in any supernumerary limbs, and histological studies of chick-quail recombinants
1974) in Albany, New York. Ectodermal
donors of quail origin were used in a few (cf. Figs. 4a and b) show no evidence that
instances in the Albany experiments. The donor mesodermal cells are adherent to
the grafted ectoderm.
embryos were incubated under standard
Host embryos were of stages 20-21. They
conditions and removed for use as hosts or
were prepared by removing albumin and
donors at the appropriate stages.
In our earlier experiments, AERs were fenestrating the egg according to the usual
prepared for grafting by isolating wing procedures (Zwilling, 1959). For grafts to
and leg buds from donor embryos of stages the dorsal side of the wing bud, ectoderm
18-24. The buds were then rinsed in cal- was removed from a narrow zone by means
cium- and magnesium-free Tyrode’s solu- of glass needles (Saunders, 1948) in order
to expose a mesodermal bed for the AER.
tion (CMF; Moscona, 1952) and incubated
in 2% trypsin (Difco 1:250) in CMF for 25
The latter was usually oriented parallel to
18
DEVELOPMENTAL
BIOLOGY
the anteroposterior
axis of the host bud on
a line joining the anterior
and posterior
junctions of the limb bud and body wall or
slightly more distal to it. In a single case
the graft was oriented parallel to the mediolateral axis of the host bud and, in another isolated instance, the wound bed was
excised to a depth of ca. 0.1 mm, rotated
180” in the horizontal
plane and replaced
before the graft was made. All grafts were
held in place by means of line glass pins
(Saunders,
1948) until healing occurred, a
matter of an hour or more.
In some cases, the orientation
of the
original anteroposterior
axis of the graft
with respect to that of the host was known.
Whether
these axes corresponded
or not
made no difference in the results,
as expected on the basis of Zwilling’s
(1956a)
experiments
and the more recent ones of
MacCabe, Errick,
and Saunders (1974).
To make grafts of AER to the ventral
side of the wing bud, the entire right wing
bud was severed at stage 18 at its junction
with the body wall and a freshly severed
left limb tip from a donor of the same stage
was grafted to the stump in reversed dorsoventral orientation.
After a lapse of 5-7
hr to permit healing of the reversed bud,
its originally ventral side was prepared to
receive the graft, as described above.
Hosts were sacrificed after a total incubation time of 11-12 days, fixed in formalin
and stained with methylene blue to reveal
cartilage (method of Lundval; Hamburger,
1960). Thereafter,
they were cleared in oil
of wintergreen
as necessary for the analysis of skeletal parts.
RESULTS
1. Morphogenetic
ary AER
Effects of a Supernumer-
Most grafts of the AER healed, but with
varying
degrees of success. All showed
considerable
shrinkage
in their longitudinal dimension
after 24 hr but, in some
cases, only a portion of the ridge adhered
permanently
to the mesoderm.
Observa-
VOLUME
50, 1976
tions in ovo were not made after 48 hr. At
that time, however, the grafts were seen to
have maintained
the orientation
in which
they were placed on the hosts and a subjacent longitudinal
swelling
of host mesoderm parallel to the long axis of the AER
was sometimes evident.
a. Grafts to the dorsal side. There were
18 hosts in which the grafted AER was
recognizable
the day after
operation.
These were allowed to continue development. When they were sacrificed,
three
showed no effects of the graft on the right
wing, and in seven cases only insignificant
excrescences
or a few extra feathers could
be recognized in the region of the right
elbow. In four of the remaining eight specimens,
supernumerary
outgrowths
of
slightly greater importance were found. In
one of these, the ulna of the host is massive and is associated with what appears to
be an incomplete supernumerary
metacarpal IV (Fig. la). In another a supernumerary
radius, connected basally to the
normal one was formed, but no supernumerary digits appeared (Fig. lb). Another
specimen shows a single digit-like
outgrowth
from the dorsal side, but is not
otherwise
identifiable.
The fourth specimen in this group shows on its dorsal side
a supernumerary
outgrowth
resembling
metacarpal
and digit III. Feather germs
are symmetrically
arranged
about this
digit and, possibly, are all of dorsal type
for, although arranged in no recognizable
dorsal pattern, they are longer than those
normally found on the ventral side of the
wing at this stage. This particular
case
resulted from the only operation in which
the supernumerary
AER
was grafted
parallel to the mediolateral
axis of the
host bud.
In contrast to these specimens are the
remaining
four, all of which show a complete set of supernumerary
hand parts
originating
from the dorsal side of the
right wing. In two of these there is also a
supernumerary
radius, the latter joining
the normal radius basally (Figs. 2a and
SAUNDERS,
GASSELINC
AND ERRICK
Supernumerary
AER
19
a
FIG. 1. Effects of grafting
a supernumerary
AER
of leg-bud
origin to the dorsal side of the right wing
bud. (a) Cleared
IO-day
wing
resulting
from
an
operation
at stage 20; there is a massive
ulna NJ)
and a supernumerary
digit
(arrow).
(b) Uncleared
wing resulting
from operation
at stage 19; specimen
illuminated
so as to highlight
the supernumerary
radius.
2b). One of the specimens resulted from
the sole operation in which the graft site
was rotated 180” with respect to the anteroposterior
and mediolateral
axes of the
wing bud. Of particular
interest in these
four cases is the fact that the supernumerary hand parts that formed under the influence of the graft have a bidorsal organization; that is, they show double sets of
remiges and major coverts mirror-twinned
in the palmar plane. A pattern of right or
left asymmetry
thus cannot be assigned to
these supernumerary
limbs.
b. Grafts to the ventral side. The foregoing results suggest that the morphogenetic
effects of a supernumerary
AER grafted to
the ventral side of the wing bud should
also be examined.
Accordingly,
in five
cases, left wing buds were grafted in reversed dorsoventral
polarity to the stump
of a severed right wing bud. These re-
FIG. 2. Uncleared
specimens
showing
extensive
duplications
resulting
from grafting,
in each case, a
supernumerary
AER of wing-bud
origin to the dorsal side of the right wing bud. (al Ten-day
wing seen
from the dorsal side after operation
at stage 18. The
supernumerary
radius Lsr) is faintly
stained
and the
position
of the normal
radius
is indicated
by an
arrow.
The supernumerary
hand (sh) rotated
counterclockwise
about its proximodistal
axis, thus obscuring one of its two sets of remiges.
The other set
is clearly
seen mirroring
the remiges
of the primary
hand. (bl In this case, the superficial
dorsal mesoderm of the host wing bud (stage 20) was reoriented
180” before receiving
the grafted
AER. A supernumerary
hand bearing
two Digits
II developed
in
reverse
anteroposterior
orientation
with respect to
the primary
hand. The secondary
hand bears two
sets of remiges,
one below the other, so that only one
set is seen in this view,
mirroring
the pattern
of
remiges
of the primary
hand.
20
DEVELOPMENTAL
BIOLOGY
ceived implants of AER on their originally
ventral sides. The resulting limb tips grew
dorsally rather than ventrally,
by virtue of
their reversed orientation,
and gave rise to
well-formed
left wings.
In all cases, supernumerary
outgrowths
appeared on the reversed limb tip in response to the grafted AER. These arose
from the originally
ventral
side. In two
instances, the outgrowth
consisted of a supernumerary
metacarpal
and digit II,
skin-covered
but lacking feather germs. In
another
instance
a skin-covered
bone,
suggestive of a reduced metacarpal III or a
metacarpal
IV, was formed. It, too, was
feather-free.
A similar
structure
with
small feather germs on both sides developed in a fourth case.
The last specimen in this group (Fig. 3)
shows a supernumerary
hand consisting of
metacarpals
III and IV and their phalanges. The anteroposterior
axis of the supernumerary
hand conforms
in polarity
to
that of the host, as determined
by the order of skeletal
parts. In contrast
to the
hand parts of the host, which show the
characteristic
dorsoventral
differentiation
VOLUME
50, 1976
of feather patterns,
both dorsal and ventral sides of the supernumerary
hand are
alike. They are essentially
free of feather
germs, corresponding
in appearance to the
ventral side of the host’s hand. Thus, they
have a biventral organization.
2. Persistence
of the Apical
Ridge
AERs of quail origin were grafted to the
dorsal side of the wing bud of chick hosts of
stages
20-21, inducing
supernumerary
outgrowths.
Hosts were sacrificed at 2 and
4 days after operation,
and the supernumerary
limb tips were fixed, sectioned,
Feulgen-stained,
and examined
for the
persistence
of quail cells, which have distinctive
nucleolar
markers
(LeDouarin,
1971). If, as suggested by some, the AER
consists of a cell population
that is constantly depleted by death and sloughing
and replaced by cells of the distally sliding
dorsal and ventral (lateral and medial) ectodermal faces, then chick cells should replace quail cells in the AER. They do not
do so, as shown in Fig. 4a and 4b, which
illustrate
persistent
quail ridges 52 and 96
hr after operation, respectively.
FIG. 3. Effects of grafting
a supernumerary
AER as seen in a disarticulated
member
that developed
from
a right wing-bud
stump bearing
a dorsoventrally
inverted
left wing tip. The principal
outgrowth
thus shows
left asymmetry.
In (a) one sees the ventral
integumentary
pattern
found by the grafted
wing tip and in (b)
the dorsal pattern
shows clearly.
Digit II of the primary
hand is poorly developed
and is visible in (bl but not
in (al. The induced
supernumerary
hand contains
an abnormal
metacarpal
III with its two phalanges
and a
reduced metacarpal
IV. These bones are in the same anteroposterior
serial order as their counterparts
in the
primary
tip. Feather
development
in the supernumerary
tip is slightly
retarded,
but the sparse pattern
of
the feather
germs is of ventral
character
as seen in both (a) and (b). The grafted
left limb tip was from a
donor of stage 19, the same age as the host; the supernumerary
AER came from the wing bud of a stage 20
donor.
SAUNDERS,
GASSELING
AND ERRICK
FIG. 4. Persistence
of grafted
quail
AER’s
on
chick host wing buds. (a) Dark field photograph
of a
Feulgen-stained
supernumerary
limb
outgrowth
capped by a quail AER. The distribution
of quail
cells in the ridge is revealed
52 hr after operation
by
the intense
light-scattering
properties
of their nucleoli. Limits
of the quail ectoderm
on each side of
the ridge are signalled
by white
lines. (b) Similar
preparation
96 hr after operation
viewed
by transmitted light.
The AER is composed
entirely
of quail
cells, as determined
by their
distinctive
nucleoli,
and quail cells also extend
to some distance
proximally in the ectoderm.
DISCUSSION
The results reported here show that the
apical ectodermal ridge of a limb bud, isolated by a combined enzymatic and microsurgical procedure from its underlying
mesoderm and from the adjoining ectoderm, can induce outgrowth of a supernumerary limb tip when grafted to the dorsal
Supernumerary
AER
21
or ventral face of a host limb bud. None of
a great variety of other operations that
affect the integrity of the ectodermal faces
of the limb bud results in the formation of
a supernumerary limb (cf., for example,
Saunders, 1958). Moreover, limb buds that
lack an AER as a result of surgical interference, disease or mutation do not form
their terminal parts (reviewed by Saunders, 1972). The present findings thus provide additional support for the concept
that the AER is an inductor of limb outThis
concept was further
growth.
strengthened recently in results reported
by Errick and Saunders (1973, 1974) who
showed that the limb-bud ectodermal hull
promotes limb outgrowth in the inside-out
configuration and that cells of the AER are
effective in outgrowth induction after enzymatic dissociation and centrifugal reaggregation.
Our findings also show that the AER
does not depend for its continued existence
on contributions of cells from the distally
sliding dorsal and ventral faces of the ectoderm, as has been reasonably suggested on
the basis of work from Amprino’s laboratory (Camosso et al., 1960; Amprino, 1965).
A chick limb tip developing under the
influence of an AER of quail origin remains tipped by quail cells. The ridge
cells, therefore, must constitute a self-sustaining population despite the fact that, as
Camossoet al. (1960) pointed out, and as is
otherwise well known (cf., for example,
Jurand, 1965), the AER is the site of considerable necrosis. The necrotic cells are
chiefly seen in the periderm layer of the
ridge, however, and are not necessarily
cells that previously degenerated within
the pseudostratified portion of the ridge
epithelium. Moreover, the columnar epithelium of the ridge does show numerous
mitotic figures (Fig. 5; see, also, Figs. l-4
of Camosso et al., 1960), although cell division is less frequent here than in the remaining limb-bud ectoderm, according to
Camosso et ~2. (1960). Nevertheless, it apparently suffices to maintain the AER.
DEVELOPMENTAL BIOLOGY
FIG. 5. Mitotic figures (m) and degenerating nuclei (d) in the AER of the chick wing bud at stage
22.
It is of further interest that supernumerary limb tips that form as a consequence of
grafting a supernumerary AER tend to
show a bidorsal or a biventral organization
accordingly as the graft is placed on the
dorsal or ventral side of the bud. The dorsoventral polarity of the AER thus does
not affect the expression of dorsoventrality
in limb parts that develop under its influence. The present results are probably best
interpreted in relation to recent reports
(MacCabe et al., 1973, 1974; Pautou and
Kieny, 1973; Errick and Saunders, 1974)to
the effect that distal limb parts that develop after reversal of the dorsoventral polarity of the ectoderm with respect to that
of the limb tip, do so with reversed dorsoventrality.
Our findings thus are part of
an ever-increasing body of evidence (cf.
also Stark and Searls, 1974) that attests to
the importance of ectoderm other than
that of the ridge for the realization of morphogenetic patterns in the limb.
The most dramatic effects of ectoderm
on limb morphogenesis are, however,
those exercised by the AER, so we now
direct attention to recent interpretations
of its developmental role. Earlier we emphasized the possibility that the chief action of the AER is to maintain the subjacent limb-bud mesoderm in a state of developmental plasticity, or lability,
such
that it can respond to as-yet-unidentified
VOLUME 50, 1976
signals that specify successively more distal positional character along the proximodistal limb-bud axis as development progresses (Rubin and Saunders, 1972; MacCabe et al., 1973). In papers from Wolpert’s
laboratory, notably in one by Summerbell
et al. (1973), it is also presumed that the
function of the ridge is to keep the mesenthyme plastic at the tip.
Wolpert’s group has proposed (cf. Summerbell et al., 1973; Summerbell and
Lewis, 1975; Lewis, 1975; Wolpert and
Lewis, 1975) that the zone of cells under
the influence of the AER constitutes a
“progress zone” wherein the generation of
successively more distal positional values
occurs independently of signals extrinsic
to the zone. According to their model, the
progress zone, through growth, serves as a
source of cells for new distal territories. As
the limb grows out and lengthens, cells
leave the progress zone proximally. These
have successively more distal positional
values the later they emerge from the
progress zone, whereas the positional
value of cells in the progress zone remains
in a state of flux. The group of cells that
first emerge from the progress zone (presumably no longer under the influence of
the apical ridge) would differentiate the
characteristics of more proximal limb levels. Later-emerging cell groups, having
“counted” more time in the progress zone,
would have generated more distal positional values and would, therefore, differentiate more distal morphological characteristics.
This model is very attractive by virtue of
its simplicity. A more complex model requires that cells in the labile zone have
their positional character imposed by signals from the zone immediately proximal
to it. Thus, Rubin and Saunders (1972) interpreted the message of the AER to the
labile zone as: “Consult the mesoderm subjacent to you and become the next level
(distally) to it.” Their interpretation arose
largely from two considerations: first, the
SAUNDERS,
GASSELING
AND
ERRICK
fact that there are signals of proximal origin, capable of transmission
through filters, that determine the distribution
and
symmetry
of terminal
limb parts (Saunders and Gasseling, 1963); second, the fact
that prospective
thigh mesoderm, grafted
subjacent to the AER of the wing bud,
forms only foot parts, as appropriate
to the
distal character of adjacent wing-bud mesoderm (Saunders et al., 1957). Sometimes
thigh feathers appeared on the forearm,
but the grafts did not form skeletal parts
of thigh or shank type, as they might have
done if they became part of a “progress
zone” and resumed “counting”
under the
influence of the AER.
A number
of papers (e.g., Saunders,
Gasseling
and Gfeller,
1958; Amprino,
1965; Camosso and Roncali, 1971) point up
the role of proximal mesodermal factors in
the determination
of regional characteristics along the anteroposterior
axis. These
have been characterized
as proximal “morphogens” by Tickle et al. (19751, which are
presumed to arise from the “Zone of Polarizing Activity,”
first described by Saunders and Gasseling (1968) and later analyzed by Balcuns et al. (1970), MacCabe et
al. (19731, and Fallon
and Crosby (1975).
That proximal factors affect the anteroposterior order of limb parts does not necessarily require, of course, that factors extrinsic to the “progress zone,” if such there
be, determine the regional differentiation
of the proximodistal
axis. There are, in
fact, several aspects of the generation
of
differentiation
along the proximodistal
axis that are not readily
explained by
either the model of Summerbell
et al. or
that of Rubin and Saunders. Why, for example, do dissociated
and reaggregated
postaxial
cells of the limb buds, recombined in an ectodermal hull, seldom make
distal limb parts, whereas similar recombinants made with preaxial limb-bud cells,
similarly
treated,
do so with high frequency (MacCabe et al., 1973; Crosby and
Fallon, 1975)? Why do recombinants
made
Supernumerary
AER
23
with dissociated
and reaggregated
cells
from proximal
wing-or
leg-bud halves
usually fail to form phalanges
whereas
those that are made with cells from distal
limb-bud
halves
almost
always
make
them (current
experiments)?
Proximal
cells are quite capable of making distal
limb parts for, as the new data presented
in this paper demonstrate,
limb buds in
ouo that receive a graft of AER proximal to
the “progress zone” can form all limb parts
of positional character distal to the graft
site. They are, therefore, capable of becoming labile under the influence of an AER.
The foregoing considerations
offer diffrculties for the acceptance of the attractive
model of Summerbell
et al.
They, also,
unfortunately,
offer little in the direction
of interpreting
the relative roles of factors,
intrinsic and extrinsic to the sub-ridge region, in controlling
the differentiation
of
positional
characters
along the proximodistal axis. Hopefully,
however, they will
be fruitful
in stimulating
further
experimentation
that will contribute
to this
problem and to the ever challenging problem of the nature of the reciprocal signals
that pass between the mesoderm of the
limb tip and the AER.
REFERENCES
R. (1965). Aspects of limb morphogenesis
in the chicken.
In “Organogenesis”
(R. L. DeHaan
and H. Ursprung,
eds.), pp. 255-281.
Holt, Rinehart and Winston,
New York.
AMPRINO,
R., and AMBROSI,
G. (1973). Experimental
analysis
of the chick embryo
limb bud growth.
Arch. de Biol. 84, 35-86.
BALCUNS,
A., GASSELING,
M. T., and SAUNDERS,
J.
W., JR. (1970).
Spatiotemporal
distribution
of a
zone that controls
antero-posterior
polarity
in the
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