Download Pattern Specification and Pattern Regulation in the Embryonic Chick

AMER. ZOOL., 22:117-129 (1982)
Pattern Specification and Pattern Regulation
in the Embryonic Chick Limb Bud1
LAURIE E. ITEN
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
SYNOPSIS. The embryonic chick limb bud is a growing organ rudiment whose undifferentiated cells give rise to a precise spatial pattern of differentiated structures. The establishment of positional values of chick limb bud cells (pattern specification) and the response of limb bud cells with established positional values to experimental perturbations
(pattern regulation) are the major topics considered in this paper. The results of recent
experiments with developing chick limb buds analyzing pattern specification and pattern
regulation are presented. These studies with the chick limb are described in light of the
postulates of a model that was originally formulated from experiments performed on
regenerating amphibian and insect appendages.
INTRODUCTION
As a chick limb bud grows out, a precise
spatial pattern of limb structures emerges.
Limb buds first appear as paired condensations of somatopleural mesoderm that
protrude from the lateral body wall at
Hamburger and Hamilton (1951) stage 16
for the wing and stage 17 for the leg. Rimming the apex of each mesodermal bulge
is an Apical Ectodermal Ridge (AER). As
the limb bud begins to grow out, the dorsal
side of the bud soon becomes rounded and
the ventral surface becomes flattened. By
stage 21 it is apparent that the posterior
half of the limb bud is growing more than
the anterior half. Overt cytodifferentiation
is first seen in the proximal third of a late
stage 22 limb bud and differentiation progresses in a proximal to distal direction. By
10 to 12 days incubation (stage 36 to 38),
the undifferentiated cells of a limb bud
have given rise to the skeletal elements,
muscles, tendons, connective tissue, feather germs and/or scales that characterize
the pattern of the adult wing or leg.
The developing chick limb is a system
chosen by many to study the process of
pattern formation. We want to know the
supracellular, cellular, and molecular aspects of how cells in a limb bud know their
physical location and how their positional
information leads to the production of the
three-dimensional pattern of limb struc1
From the Symposium on Principles and Problems
of Pattern Formation in Animals presented at the An-
nual Meeting of the American Society of Zoologists,
27-30 December 1980, at Seattle, Washington.
tures. Much of our work is based on the
concept of positional information first proposed by Driesch (1908) and still widely
accepted by developmental biologists. The
concept of positional information is that
during development cells acquire specific
fates according to their physical location in
the embryo. The positional information
theory of Wolpert (1969, 1971) is that cells
have their positional values (positional information) specified and then they interpret their positional values with the appropriate cytodifferentiation. Cells which
have their positional values established
with respect to the same coordinate system
constitute a developmental field. The early
embryo of many vertebrates is considered
to be a single (primary) field and as embryogenesis proceeds, secondary fields can
be operationally identified in the embryo
by their self differentiation and regulative
capacities (French etai, 1976). Developing
chick limb buds are secondary fields. For
the sake of clarity in the discussion that
follows, pattern specification is defined as the
initial establishment of positional values of
cells in a field and pattern regulation as the
process whereby cells in a developmental
field with established positional values respond to various experimental perturbations.
Some of the types of analysis we and others have used to study pattern specification
and pattern regulation in the developing
chick limb are the following. (1) Analysis
of the ability of the chick limb bud to compensate (regulate) after different portions
117
118
LAURIE E. ITEN
of the limb bud are removed or added. (2)
Analysis of the pattern of limb structures
formed after juxtaposing normally nonadjacent limb bud cells. (3) Analysis of the
relationship of pattern specification and
pattern regulation to cell, tissue, and organ
growth. (4) Analysis of the portion of the
limb pattern that progeny of the marked
limb bud cells form. (5) Analysis of limb
mutants which show abnormal spatial patterns of differentiation. All these types of
anlayses have been used to formulate
models to account for the normal pattern
of limb structures formed during development. Many of the limb pattern formation models have been instrumental in
stimulating a great deal of research, all to
the benefit of those working in the field.
No attempt will be made to present an exhaustive review of what all these types of
analyses tell us about pattern specification
and pattern regulation in developing chick
limbs, nor will I discuss the merits or demerits of current models. Instead, I will
focus on those experimental studies and
hypotheses dealing with the formation of
supernumerary limbs and limb structures
and what they tell us about pattern specification and pattern regulation.
DISHARMONIC AXIAL REVERSALS
Supernumerary limbs or limb structures
form after disharmonic axial reversals of
a chick limb bud on its stump. Saunders
et al. (1958) were the first to show that
when the distal third of a left wing bud is
grafted to the contralateral limb stump
with the antero-posterior axis of graft and
stump misaligned, supernumerary limb
structures usually develop; however, when
similar grafts are performed, but with the
dorso-ventral axis opposed, supernumerary skeletal elements do not form, although supernumerary integumentary
structures form and supernumerary muscles are found at the graft junction (Iten
and Majors, unpublished results). When
the distal third of a right limb bud is reoriented on its base after 180° rotation
about its proximo-distal axis, supernumerary limbs form (Saunders et al, 1958; Amprino and Camosso, 1958, 1959; Saunders
and Gasseling, 1959; Amprino, 1968; Ca-
mosso and Roncali, 1971). In all these situations where supernumerary limbs form,
corresponding supernumerary outgrowths covered by an AER can be detected one to two days after the operation.
THE ZONE OF POLARIZING ACTIVITY AND
ITS MORPHOGEN(S)
Saunders and co-workers were the first
to propose that the stimulation of supernumerary limb outgrowths is due to juxtaposing posterior limb bud cells next to
anterior cells (Saunders and Gasseling,
1968). These posterior limb bud cells were
named the Zone of Polarizing Activity
(ZPA) (Balcuns et al, 1970) and the ZPA
of a limb bud was operationally defined as
those posterior cells that have the capacity
of stimulating the formation of polarized
supernumerary limb structures when
transplanted to an anterior position in a
host limb bud (A. B. MacCabe et al, 1973).
For example, when ZPA tissue is grafted
to the anterior edge of a host's right wing
bud, the AER posterior to the graft becomes thickened, the limb bud widens, and
supernumerary left wing structures arise
from the outgrowth of the host's anterior
wing tissue; the pattern of structures in a
resulting limb is symmetric about its long
axis. Other properties of the ZPA include
its capacity to polarize limb structures
formed by dissociated limb bud mesenchyme in a limb bud ectodermal jacket (J.
A. MacCabe et al, 1973). If the ZPA is dissociated and coaggregated with dissociated
anterior or posterior limb bud mesenchyme, it inhibits limb outgrowth and the
formation of distal structures (Crosby and
Fallon, 1975). The interaction between the
ZPA and host limb tissue is nonspecific
among different species of birds, between
birds and reptiles, or birds and mammals
(Balcuns et al, 1970; Saunders and Gasseling, 1968; Saunders, 1972; MacCabe
and Parker, 1976a; Tickle et al, 1976; Fallon and Crosby, 1977).
A model has been formulated that assigns to the ZPA the function of specifying
limb bud cells' antero-posterior positional
coordinates (Tickle et al, 1975; Summerbell and Fickle, 1977). It is postulated that
PATTERN SPECIFICATION AND REGULATION
the ZPA is a localized source of a morphogen at the posterior margin of a limb bud.
The morphogen diffuses through the rest
of the limb bud where it is broken down,
resulting in an exponential concentration
profile of morphogen with the maximum
concentration at the ZPA. A cell's anteroposterior positional coordinate is specified
by the concentration of morphogen to
which it is exposed while in the progress
zone (mesenchyme cells approximately
300 /Am subjacent to the AER). The progress zone is also where it is proposed that
cells acquire their proximo-distal positional coordinate by measuring the time they
spend in this zone; cells leaving the progress zone early have proximal positional
values and cells leaving later have more
distal positional values (Summerbell et al.,
1973; Summerbell and Lewis, 1975). Limb
bud cells subsequently interpret their positional value which leads to the anteroposterior and proximo-distal pattern of
differentiated limb structures. Therefore,
a diffusible signal from the ZPA specifies
a cell's antero-posterior positional coordinate and an autonomous timing (counting)
mechanism specifies a cell's proximo-distal
coordinate.
According to ZPA/Progress Zone model,
if an additional ZPA is transplanted to an
anterior position in a host limb bud, then
anterior cells in the progress zone that
were previously exposed to a low concentration of morphogen are now exposed to
a high concentration of morphogen.
These cells have their antero-posterior positional value changed such that they will
form posterior rather than anterior limb
structures; the proximo-distal level where
this change will occur corresponds to the
proximo-distal positional values of cells
leaving the progress zone (Summerbell,
1974a). In order to account for the growth
in width of a limb bud after a ZPA grafting
operation, it was originally proposed that
the steepness of the morphogen gradient,
rather than the local absolute concentration of morphogen, controls the rate of cell
proliferation. More recently, it has been
postulated that there is another signal
coming from the ZPA and it is a growthpromoting signal that influences the cell
119
cycle of limb bud cells (Cooke and Summerbell, 1980).
Attempts to isolate the morphogen specifying a cell's antero-posterior positional
value or the signal influencing the growth
of limb bud cells have been unsuccessful
thus far. MacCabe and Parker (1975) have
shown that isolated ZPA mesenchyme can
maintain an AER in vitro when either in
contact or separated from the AER. Using
this in vitro bioassay for maintenance of an
AER, they find high activity with posterior
mesoderm, low activity with central mesoderm, and no activity with anterior mesoderm (MacCabe and Parker, 19766). From
these results they propose that there is
some factor being generated from the
ZPA, but it remains to be seen whether this
factor has anything to do with assigning
positional values to limb bud cells or influencing the cell cycle of limb cells.
LOCAL CELL INTERACTIONS AND THE
POLAR COORDINATE MODEL
The polar coordinate model of French
et al. (1976) was formulated from studies
on regenerating amphibian and insect appendages. This model, like the ZPA/Progress Zone model, is based on the concept
of positional information. It is postulated
that cells in a secondary field have their
positional values specified in a polar coordinate system. One component of a cell's
positional information is a value corresponding to position on a circle, and the
second to position on a radius. One rule of
cellular behavior proposed by this model
is that when cells with normally nonadjacent positional values are next to each
other, growth occurs until cells with intermediate positional values have been
intercalated and the discontinuity is eliminated. The other rule of this model deals
with the specification of cells with progressively more distal positional values. The
generation of cells with more distal values
(distal transformation) is seen as the result
of cells with different circumferential positional values interacting with each other
and then intercalating (Bryant et al., 1981).
Both rules of the model propose that pattern regulation (intercalation) and pattern
120
LAURIE E. ITEM
TPH
TPH
EMU
EMU
Anc
Anc
FCU
FDP
FDP
FDS
EML
EML
EMB
EMU
EMU
UMD
specification (distal transformation) are
the result of local cell interactions.
When the first experimental results with
regenerating amphibian limbs were described with the polar coordinate model,
it seemed as though there were results with
developing chick limbs that could also be
described with this model (Bryant and
Iten, 1976). And more important, several
predictions based on the postulates of this
model could be experimentally tested with
the developing chick limb. What follows is
a description of some of the things we have
learned by using this hypothesis to study
pattern specification and pattern regulation in the embryonic chick limb bud.
JUXTAPOSING NORMALLY NONADJACENT
LIMB BUD CELLS AND THE FORMATION OF
SUPERNUMERARY LIMB STRUCTURES
According to the polar coordinate model, when normally nonadjacent chick limb
bud cells are juxtaposed in grafting operations, cells with discontinuous positional
values are next to each other. Such a situation should result in extra growth by
donor and host cells and Cooke and Summerbell (1980) show that there is such a
stimulation of cell division (see their Figure 3). With the polar coordinate model,
it is proposed that this extra growth represents cells with positional values that
UMD
FDP FDP
FIG. 1. Camera lucida drawings of three sections of
a resulting wing after a wedge of posterior edge stage
21 right quail (Cotumix coturnix japonica) wing bud
tissue (adjacent to somites 19 and 20) was transplanted to an anterior slit made adjacent to the junction
of somites 16 and 17 of a right stage 21 chick wing
bud. The proximo-distal and dorso-ventral polarity
of donor and host tissue were matched and the distal
edge of the graft and host were aligned with each
other. Resulting wings were fixed in Zenker's at 10
to 12 days incubation. Serial 7 /xm cross sections of
wings were stained with the Feulgen nuclear reaction.
Quail cells were identified by their large heterochromatic nucleoli. The top section is one through the
upper arm, the middle section is one through the
forearm, and the bottom section is one through the
hand of a resulting wing. The embryo's axes are noted in the top section with the following abbreviations:
A, anterior; P, posterior; D, dorsal; V, ventral. Where
quail cells are seen in these sections is indicated by
the stippling. The abbreviations for the skeletal elements and muscles in these sections and the sections
shown in Figure 2 are the following: H, humerus; R,
radius; U, ulna; 2, 3, and 4, metacarpals of digits 2,
3, and 4; M5, metacarpal of digit 5; AbM, abductor
medius; Adi, adductor indicis; Anc, anconeus; Bic,
biceps; Del, deltoid; EDC, extensor digitorum communis; EIL, extensor indicis longus; EMB, extensor
medius brevis; EMR, extensor metacarpi radialis;
EML, extensor medius longus; EMU, extensor metacarpi ulnaris; FCU, flexor carpi ulnaris; FDP, flexor
digitorum profundis; FDS, flexor digitorum superficialis; FI, flexor indicis; LD, latissimus dorsi; TP,
tensor propatagii; TPH, triceps pars humeralis; TPS,
triceps pars scapularis; UMD, ulnimetacarpalis dorsalis; L'MV, ulnimetacarpalis ventralis. The scale bar
at the bottom right represents 1 mm.
PATTERN SPECIFICATION AND REGULATION
Del
EDC
EML\ EMU
EIL \ \
/
/Anc
EMR
FDP
UMV FDS
EMB
/
FCU
EML
EMU
UMD
Fie. 2. Camera lucida drawings of three sections of
a resulting wing after the same posterior wedge of
quail wing bud tissue described in Figure 1 was transplanted to a posterior edge slit made adjacent to the
junction of somites 19 and 20 of a right stage 21 chick
wing bud. The proximo-distal and dorso-ventral polarity of donor and host tissue were matched and the
distal edge of the graft and host were aligned with
each other. Resulting wings were fixed, sectioned,
and stained as described in Figure 1. As in Figure 1,
the top section is one through the upper arm, the
middle section is one through the forearm, and the
bottom section is one through the hand of a resulting
121
eliminate the discontinuity caused by the
grafting operation. Donor and host cells
that are intercalated are those that make
up the extra structures formed. In Figure
1, we show that donor posterior quail wing
bud cells grafted to an anterior host site in
a chick wing bud contribute to the extra
(duplicated) limb structures formed. For
comparison, Figure 2 shows where quail
cells are found when posterior quail wing
bud tissue is grafted to a posterior host
site, i.e., grafted to the same position as its
position of origin. The legends to these
two figures give the details of the operations performed. Cells from host tissue
also contribute to the extra structures
formed and a detailed description of the
contribution of donor and host limb bud
cells to the formation of supernumerary
limb structures will be presented elsewhere
(Iten, in preparation).
With the polar coordinate model,
whether supernumerary limb structures
form and more importantly, what extra
structures form should depend on the positional disparity created by a transplantation operation. The first results we obtained that support this prediction were
with operations where anterior or mid
limb bud tissue was transplanted to more
posterior positions in a host wing bud (Iten
and Murphy, 1980). Whether extra structures formed and what extra structures
formed depended on the position of origin
of donor tissue, the antero-posterior position donor tissue was put in a host wing
bud, and the dorso-ventral axial alignment
or misalignment of donor and host tissue.
Two subsequent studies where different
portions of posterior wing bud tissue were
transplanted to more anterior positions in
a host wing bud also support this prediction of the model (Javois and Iten, 1981;
Javois et al, 1981). These later two studies
wing. Where quail cells are seen is indicated by the
stippling. Abbreviations and scale bar as in Figure 1.
It is important to note here that even though quail
wing bud tissue was added to a host chick wing bud,
wings with a normal pattern and complement of skeletal elements and muscles result following such control transplantation operations as described here.
122
LAURIE E. ITEN
_J
1
FIG. 3. At the top is a dorsal view outline of a stage
21 right wing bud and adjacent somites drawing the
angle of cut made to remove the limb bud and a
dorsal view outline after 180° rotation of the severed
wing bud on its base. The distance from the center
of one somite to the center of the next is approximately 300 fum and the antero-posterior length of a
wing bud at its base is about 1 mm. Below is a dorsal
view of the skeletal pattern of two examples of wings
resulting from this rotation operation. One wing has
three forearm skeletal elements and an anterior to
posterior digital sequence of 2, 3, 4 (below and not
clearly visible in this photograph), 3, 3, 4. The other
wing has four forearm skeletal elements and an anterior to posterior digital sequence of 4, distally duplicated 3, 4. A normal skeletal pattern is shown in
Figure of.
illustrate the importance of examining the
muscle and integumentary pattern along
with the skeletal pattern of resulting limbs
when assessing the effects of placing normally nonadjacent limb bud cells next to
each other.
All the above studies showing that supernumerary limb structures form after
juxtaposing normally nonadjacent limb
bud cells were graft operations where an
antero-posterior positional disparity between donor and host cells was created.
With the polar coorinate model, we would
predict that a dorso-ventral positional disparity between donor and host limb bud
cells, without an associated antero-posterior disparity, should also result in the formation of extra limb structures. We have
completed a systematic and detailed study
showing that dorso-ventral opposition of
wing bud tissue alone results in the formation of supernumerary muscles (Javois
and Iten, 1980, 1982).
Saunders et al. (1958), in their study
where they performed limb bud axial misalignment operations, suggested that the
formation of what they called "triplicate"
rather than the usual "duplicate" limbs
could be due to the manner a limb bud tip
was severed from its base. To remove a
limb bud tip, the cut was usually made parallel to the base of the limb bud. We have
rotated wing buds 180° on their stump
where the cut to remove the wing bud was
made perpendicular to the future caudal
direction of outgrowth of the wing bud
(Fig. 3). One to two days after doing such
a rotation operation, three areas of limb
bud outgrowth could often be observed.
One outgrowth appears to originate from
anterior stump tissue next to the rotated
limb bud, another appears to be the
growth of the rotated limb bud, and the
third is near the posterior graft junction.
Of the 13 wings resulting from this operation, 10 would be categorized as triplicate
limbs. Two examples are shown in Figure
3. I will not give a lengthy description here
of how the polar coordinate model describes the asymmetry (handedness) of the
supernumerary wings and wing structures
formed following 180° rotation of a limb
on its stump. Instead, I will simply state
PATTERN SPECIFICATION AND REGULATION
that the handedness of the anterior and
posterior supernumerary structures appears to be in accordance with the predictions of the polar coordinate model. A subsequent paper will present a detailed
description of the handedness of supernumerary limbs formed as a result of these
and other transplantation operations (Javois and Iten, in preparation).
The high frequency of triplicate wings
resulting after 180° rotation of a limb bud
that had been severed from its base at an
angle lead us to reinvestigate whether or
not limbs with duplicated (supernumerary) structures result if posterior wing bud
tissue is removed before the tip is rotated
on its base. Fallon and Crosby (1975) reported that duplicated structures do not
result after such an operation, but their
diagrams indicate that they made a cut
parallel to the base of the bud to remove
the tip. The surgical operation we performed is shown in Figure 4. Two regions
of limb bud outgrowth are typically seen
1-2 days following this operation: one appears to originate from anterior stump tissue next to the rotated tip and the other
appears to be the growing rotated tip. As
can be seen from the two examples of
wings resulting from this operation shown
in Figure 4, an extra forearm skeletal element forms, but clearly identifiable duplicated (supernumerary) digits do not form.
While these resulting wings might not be
as spectacular as those obtained when a
whole limb bud tip is rotated on its stump,
they still show that juxtaposing normally
nonadjacent limb bud cells results in the
formation of extra (duplicated) limb structures.
DISTAL TRANSFORMATION AND
PATTERN SPECIFICATION
With the polar coordinate model, there
should be the progressive specification of
cells with more distal values (distal transformation) during the outgrowth of a secondary field. There is a great deal of experimental evidence suggesting that there
is a sequential specification of chick limb
bud cells with more distal positional values
during limb bud outgrowth. Saunders
(1948) was the first to show that removing
FIG. 4. At the top is a dorsal view outline of a stage
21 right wing bud and adjacent somites after posterior limb bud tissue has been removed and a dorsal
view outline after the remaining wing bud has been
severed from its base at an angle and rotated 180°.
Below is a dorsal view of the skeletal pattern of two
examples of wings resulting from this rotation operation. Both of these wings have an extra forearm
skeletal element and three digits, some of which are
unidentifiable. A normal skeletal pattern is shown in
Figure of.
the AER of an early stage wing bud results
in a truncated wing with missing distal
structures; limb truncation at more distal
levels results when the AER is removed
from later stage wing buds. Summerbell
(19746) later substantiated these results
and expanded the range of stages when
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LAURIE E. ITEX
TABLE 1. Results of removing the entire apical ectodermal ridge (AER) from different stage right wing buds.
Skeletal elements formed
Stage"
AER
removed
18
19
20
21
22
23
Shoulder
girdle
Partial"
humerus
Humerus
1
4 (49%)c
14 (67%)
17(76%)
3
1
2
6
12 (99%)
2
Humerus +
partial ulna
Humerus +
ulna
3
23 (37%)
2
2
3
Humerus 4ulna +
partial radius
9 (64%)
a
Length : width ratios of Hamburger and Hamilton (1951) were used to stage wing buds. The L:W ratios
used for each stage are the following: stage 18, >6; stage 19, 6-4; stage 20, 3.9-3; stage 21, 2.9-2.3; stage 22,
2.2-1.5; stage 23, 1.4-1.
b
A partial skeletal element is one whose length is less than 95% of the length of the corresponding element
of the embryo's contralateral left wing.
c
Percentages in parentheses are the mean percent length of the most distal skeletal element of resulting
right wings for the majority category. The percent length of a right skeletal element is
length of right element
x 100.
length of corresponding left element
the AER of a wing bud was removed. Removing the AER from a limb bud is seen
as stopping distal transformation and
these results have been interpreted by
many to mean that at early stages, limb
bud cells with proximal positional values
are specified and as development proceeds, cells with more distal positional values are added (Summerbell et al., 1973;
Summerbell, 19746; Summerbell and Lewis, 1975; Wolpert et al, 1979).
What might be called a "specification
map" was made from the AER removal
operations of Summerbell (19746) (Figure
5 of Summerbell and Lewis, 1975). From
this map we could say that a stage 18 wing
bud only has cells specified to form a humerus; cells specified to form a complete
radius and ulna are added by stage 21, a
wrist by stage 25, and digits by stage 28.
From the description and figures of resulting truncated wings, it appears that the
proximo-distal and antero-posterior specification of cells are in concert. For example, a wing truncated at the level of the
forearm has an anterior radius and a posterior ulna. But Figure 3 of Summerbell
(19746) implies that truncated wings with
only one forearm skeletal element can result.
If there is a high frequency of limbs
truncated at the level of the forearm with
only one forearm skeletal element and it
is always the same element then we would
conclude that cells with the positional values to form one element are specified before cells specified to form the other. We
examined this possibility by doing yet
another series of AER removal operations.
First we began by removing the complete
AER from stage 19, 20, and 21 wing buds
thinking that we would get limbs truncated
at the level of the forearm, as Summerbell
(19746) did. But there must be some difference in the way we and Summerbell determine the stage of a wing bud because
removing the AER from what we call stage
19 through 21 wing buds results in wings
with a partial or complete humerus (Table
1; Fig. 5a, 5b, 5c). When we removed the
AER from stage 22 and 23 wing buds,
limbs with a complete humerus and a
partial or complete ulna resulted (Table 1;
Fig. 5d, 5e). The fact that the forearm element was an ulna and not a radius was
confirmed by examining the pattern of
muscles surrounding the single forearm
element from serial cross sections of several of these limbs. No wings with a humerus and only a radius have been obtained. We interpret these results to mean
that cells are specified to form the posterior ulna before the anterior radius.
There is some controversy as to whether
there is a progressive (sequential) specification of the limb pattern or whether the
PATTERN SPECIFICATION AND REGULATION
125
FIG. 5. Dorsal view of the skeletal pattern of wings resulting after the entire AER of different stage right
wing buds was removed, a. AER removed at stage 19. Wing has a partial proximal humerus. b. AER removed
at stage 20. Wing has a partial proximal humerus. c. AER removed at stage 21. Wing has a complete humerus.
d. AER removed at stage 22. Wing has a complete humerus and a partial proximal ulna. e. AER removed at
stage 23. Wing has a complete humerus and ulna and a partial proximal radius. / Normal wing skeletal
pattern with a proximal humerus, forearm with an anterior radius and posterior ulna and digits 2, 3, and 4
(anterior to posterior).
entire limb pattern is specified in early
stage limb bud cells and subsequent development is the expansion of this pattern.
Stark and Searls (1973) put labelled limb
bud cells in different positions in different
stage unlabelled wing buds. Two to three
days later when the cartilage pattern was
apparent in histological sections, the de-
veloping skeletal elements with labelled
cells were identified. Their results show
that cells forming the skeletal elements of
all three limb segments can be "mapped"
in as early as a stage 18 wing bud. From
these results, they conclude that cells representing the three limb segments are
present in early stage wing buds and that
126
LAURIE E. ITEN
subsequent development only requires po- ential positional values that eliminate the
larized outgrowth, changes in cell growth, discontinuity are intercalated via the shortcell death, and differentiation. In other est of the two circular routes. Distal transwords, their presumptive fate map is formation will commence and will be proequivalent to a specification map. We and portional to the number of circumferential
others would not make such an equation positional values present or intercalated
(Wolpert et al., 1979). No one will dispute (Bryant et al, 1981).
the fact that all or almost all cells of stage
A prediction that can be made from this
18 through 22 wing buds are incorporat- polar coordinate model description of
ing tritiated thymidine (Janners and what happens after normally nonadjacent
Searls, 1970; Summerbell and Lewis, cells are placed next to each other is that
1975) and that the progeny of cells of early circumferential intercalation should occur
stage wing buds will give rise to structures even when there is no distal transformaof all three limb segments. Marking limb tion. Summerbell (1974) presents some rebud cells and seeing what structures their sults suggesting that circumferential interprogeny make tell us nothing about the calation does not occur without associated
specification of those originally marked distal transformation. He grafted postecells.
rior wing bud tissue to an anterior host site
The validity of a specification map for of wing buds that had had the anterior half
the developing chick limb constructed of their AER removed. He reports that
from the AER removal results is also ques- wings without duplicated (supernumerary)
tioned. A major criticsm is that removing structures result indicating that no circumthe AER from a wing bud does not abrupt- ferential intercalation had occurred. We
ly stop cell division of limb bud cells (Jan- have repeated this operation and analyzed
ners and Searls, 1971), but there is a re- the results in more detail.
duction in the rate of outgrowth of a wing
First we determined what wing strucbud without its AER (Summerbell, 19746). tures form after the anterior half of the
Removal of the AER does cause some cell AER was removed from stage 21 wing
death in the denuded distal 100-200 /xm buds (Fig. 6). Seven of the eight resulting
of limb bud mesenchyme (Janners and wings were missing their anterior radius
Searls, 1971; Cairns, 1975), but not all dis- and most of these (5/7) were also missing
tal cells die (see Figure 2 of Saunders, their digit 2 (Fig. 6); there was one result1977). Even with these reservations about ing wing with a complete complement of
the significance of the AER removal re- skeletal elements. Saunders (1948) also results, we still support the assertion that ported that wings with a missing radius
they show that there is a progressive spec- and digit 2 result following such an AER
ification of the limb pattern during devel- removal operation. The pattern of musopment.
cles, as determined from serial cross sections of some of these resulting wings, was
DISTAL TRANSFORMATION AND
also examined. Proximally, the pattern of
PATTERN REGULATION
upper arm muscles is normal, but in the
When we graft posterior limb bud tissue distal upper arm, the pattern is abnormal,
to an anterior host site, we are placing cells i.e., anterior muscles fade away before
with normally nonadjacent circumferential they make an insertion on a skeletal elepositional values next to each other ac- ment or they make an abnormal insertion
cording to the polar coordinate model. We on the humerus. In the forearm and hand,
can see that the AER posterior to the graft anterior muscles are missing.
thickens, the limb bud widens and a wing
Next, we transplanted posterior wing
with a duplicated humerus, a forearm with bud tissue to an anterior host site of stage
two ulnas and a hand with an anterior to 21 wing buds that did not have their anposterior digital sequences of 4, 3, 2, 3, 4 terior AER (Fig. 7). The host wing buds
typically results (Javois and Iten, 1981). It did not widen and the wings that resulted
is hypothesized that cells with circumfer- had a duplicated proximal portion of their
PATTERN SPECIFICATION AND REGULATION
12?
FIG. 6. At the top is a dorsal view outline of a
stage 21 right wing bud and adjacent somites after
the anterior half of the AER is removed. Below is a
dorsal view of the skeletal pattern of a typical wing
resulting from this AER removal operation that is
missing its radius and digit 2.
humerus (6 of 9 wings), a forearm with
only a posterior ulna (8 of 9 wings), and a
hand consisting of digits 3 and 4 in all cases
(Fig. 7). One resulting wing had a forearm
consisting of a partial proximal anterior
ulna and a complete posterior ulna. The
muscle pattern of some of these resulting
wings was also examined. In the upper
arm, there are extra muscles proximally,
but they fade away before they make a distal insertion or they make an abnormal insertion on the humerus. In the forearm
and hand, anterior muscles are missing.
The formation of extra proximal limb
structures following this transplantation
operation suggests to us that circumferential intercalation does occur without associated distal transformation.
CONCLUSION
A brief description of some of our experimental studies where we have used the
FIG. 7. At the top are dorsal view outlines of donor
and host stage 21 right wing buds and their adjacent
somites showing the position of origin of the donor
posterior wedge and the anterior position where it was
added to a host that had had the anterior half of its
AER removed. Below is a dorsal view of the skeletal
pattern of a typical wing resulting from this transplantation operation that has a duplicated proximal
humerus, a forearm with only a posterior ulna and
a hand with digits 3 and 4.
postulates of the polar coordinate model
to further our understanding of pattern
specification and pattern regulation in developing chick limbs has been presented.
Some of these studies are reinvestigations
and reinterpretations of what others in the
field have done. At times, we have obtained slightly different results because of
variations in the operational design or be-
128
LAURIE E. ITEN
cause of a more detailed analysis of the
experimental results. If experiments are
repeated or modified in light of a new or
different model and new information is
obtained, then the model has served a
worthwhile function.
We have chosen to use the polar coordinate model to study the process of pattern formation in the developing chick
limb because at this time, it is a viable
working hypothesis. Whether or not this
model will be able to accurately describe
the emergence of spatial patterns of cellular differentiation in the embryonic
chick limb bud is inconsequential. What is
important is that it gives us another vantage point from which to examine chick
limb pattern formation.
ACKNOWLEDGMENTS
in Notophthalmus viridescens and a new interpre-
tation of their formation. Develop. Biol. 50:212234.
Cairns, J. M. 1975. The function of the ectodermal
apical ridge and distinctive characteristics of adjacent distal mesoderm in the avian wing-bud. J.
Embryol. Exp. Morph. 34:155-169.
Camosso, M. and L. Roncali. 1971. Modificazioni
organogenetiche di territori dell'ablozzo dell'ala
in condizioni varie di trapianto. Soc. Ital. Biol.
Sperimentale 47:673-675.
Cooke, J. and D. Summerbell. 1980. Cell cycle and
experimental pattern duplication in the chick
wing during embryonic development. Nature
287:697-701.
Crosby, G. M. and J. F. Fallon. 1975. Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre- or postaxial
wing bud mesoderm. Develop. Biol. 46:28-39.
Driesch, H. 1908. The science and philosophy of the or-
ganism. Black, London.
Fallon, J. F. and G. M. Crosby. 1975. The relationship of the zone of polarizing activity to supernumerary limb formation (twinning) in the chick
wing bud. Develop. Biol. 43:24-34.
Fallon, J. F. and G. M. Crosby. 1977. Polarizing zone
activity in limb buds of amniotes. In D. A. Ede,
J. R. Hinchliffe, and M. Bells (eds.), Vertebrate
A number of co-workers contributed to
the research reported herein and I want
to express my gratitude to Lorette C. Javois, Glenn N. Major, Joan B. McDermott,
limb and somite morphogenesis, pp. 55-69. Cambridge University Press, Cambridge.
Kirk P. Morey, and Douglas J. Murphy for
all their help. This research was supported French, V., P. J. Bryant, and S. V. Bryant. 1976.
Pattern regulation in epimorphic fields. Science
by Grant Number PCM 77-08404 A01,
193:969-981.
awarded by the National Science Founda- Hamburger, V. and H. L. Hamilton. 1951. A series
tion. I wish to thank Drs. Susan V. Bryant
of normal stages in the development of the chick
embryo. J. Morphol. 88:49-92.
and Nigel Holder for their invitation to
participate in this symposium they have Iten, L. E. and D. J. Murphy. 1980. Pattern regulation in the embryonic chick limb: Supernuorganized on the Principles and Problems
merary limb formation with anterior (non-ZPA)
of Pattern Formation in Animals.
limb bud tissue. Develop. Biol. 75:373-385.
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