/ . Embryol. exp. Morph. Vol. 69, pp. 7-36, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Control of pattern formation
in urodele limb ontogeny: a review and a
hypothesis
By DAVID L. STOCUM 1 AND JOHN F. FALLON 2
From the Department of Genetics and Development
University of Illinois
and Department of Anatomy, University of Wisconsin
SUMMARY
From a review of the literature, the hypothesis is advanced that the forelimb region of the
urodele embryo acquires its transverse axial polarity and pattern by the action of posterior
and dorsal polarizing regions. The anterior-posterior and dorsal-ventral axes are determined
simultaneously and their determination is a prerequisite for proximal-distal outgrowth.
Outgrowth of the limb bud is accompanied by the generation, between proximal and distal
boundaries, of a set of positional values specifying proximal-distal axial polarity and pattern.
The proximal boundary is the initial positional value carried by the cells of the limb
area. The distal boundary is imposed upon the outermost layer of limb disc cells by the
overlying ectoderm.
INTRODUCTION
Developmental biologists in the 20th century have been confronted with two
major problems of gene regulation whose domains can be arbitrarily separated
at the surface of the cell. The first problem is to understand the sequence of
intracellular events that result in a pattern of synthetic activity directed by the
genome and associated with a specific differentiation. The second problem is to
understand the intercellular interactions which specify regional patterns of gene
activity, and thus the regional patterns of differentiation which we recognize
as tissues and organs (pattern formation). The problem of how pattern is
determined, which historically has proven to be so intractable, is currently a
subject of renewed interest, largely because of the detailed theoretical analyses
of Wolpert (1969, 1971), French, Bryant & Bryant (1976) and Bryant, French &
Bryant (1981).
One of the first and most productive systems for the analysis of pattern
formation and regulation was the developing urodele forelimb. The goal of
1
Author's address: 515 Morrill Hall, 505 South Goodwin Ave., Department of Genetics
and Development, University of Illinois, Urbana, Illinois 61801, U.S.A.
8
Author's address: Department of Anatomy, University of Wisconsin, Madison, WI 53706
U.S.A.
8
D. L. STOCUM AND J. F. FALLON
investigators working with this system has been to understand the processes
underlying (1) the initial segregation of the limb mesoderm from the somatic
lateral plate mesoderm, and (2) the development of axial polarity and tissue
pattern of the limb mesoderm, which is closely linked to outgrowth of the
limb from the body wall. Little is known about the first process, but a large body
of experimental data has been accumulated on the second. From these data has
emerged the view that the limb rudiment is a harmonious, equipotential system
whose three cardinal axes of asymmetry are determined in the following
sequence: anterior-posterior (AP), dorsal-ventral (DV), and proximal-distal
(PD) (Harrison, 1918, Swett, 1937). The mechanisms of polarization and
pattern formation, however, remain unknown. In the present paper, we (1)
review the literature on urodele limb development, (2) challenge the classical
interpretation of the data relating to axial determination, and (3) propose a
hypothesis of axial polarization and pattern development during limb ontogeny
which can be integrated with limb regeneration.
Location and map of the forelimb area
The presumptive forelimb rudiment has been located and mapped in several
species of Ambystoma and Triturus by extirpation experiments (Detwiler, 1918;
Harrison, 1918; Suzuki, 1928; Rotmann, 1931; Takaya, 1941), vital dye
tracking (Detwiler, 1918,1929,1933; Swett, 1923), and construction ofchimaeric
limb rudiments (Schwind, 1928, 1931, 1932). At early gastrula, the presumptive
forelimb ectoderm is located bilaterally within the presumptive skin ectoderm
region, about 130° anterior to the crescent-shaped blastopore. The presumptive
forelimb mesoderm is located bilaterally in the lateral plate mesoderm, a few
degrees lateral to the blastopore (Rotman, 1931; Detwiler, 1933). The morphogenetic movements of gastrulation position the prospective limb mesoderm
under its corresponding ectoderm. By the medullary fold stage, the limb
rudiment is located just posterior to a line dividing the anterior two-thirds and
posterior one-third of the embryo (Detwiler, 1918, 1929). At the tailbud stage,
the limb rudiment has been classically defined as a 3-5-somite-wide disc whose
AP diameter stretches from the anterior border of somite 3 to the middle of
somite 6, with its dorsal border overlapping the pronephros (Detwiler, 1918;
Harrison, 1918; Takaya, 1941). However, as described below, the actual
prospective limb tissue occupies only a part of this disc.
Figure 1 is a map of the Harrison stage-29 forelimb area of Ambystoma maculatum(punctatum) identifying the regions which will form the free limb and girdle
and also the non-limb regions which are capable of regulating to form limb
and girdle if prospective limb cells are removed. We stress two points about this
map. First, the inner large circle of tissue, 3-5-somites wide, has been classically
denned as the 'limb disc'. This is the area that has been transplanted in most
experiments investigating the problem of axial determination in limb ontogeny.
Second, only slightly more than the anterior half of this disc actually represents
Urodele limb ontogeny
Fig. 1. Map of the presumptive right forelimb and surrounding areas of the Harrison
stage-29 Ambystoma maculatum embryo, constructed from the data of Detwiler
(1918), Harrison (1921, 1925), Swett (1923), and Schwind (1931). The shoulder
girdle develops from the region marked by hatching, while the free limb (FL) is
derived from a 2-2-5 somite-wide area (clear circle). The AP and DV axes of the free
limb are rotated 30-45° (clockwise for the right limb, counterclockwise for the left)
from the corresponding body axes. The stippled area is peribrachial flank tissue
(PBF) that does not normally take part in forelimb development. The inner large
circle (3-5-somite diameter) encompasses the classical limb disc that has been used in
transplantation experiments, even though much of its posterior tissue is not prospective limb. The outer large circle (5-somite diameter) represents the maximum size
of the region that can regulate to form limb after actual presumptive limb tissue is
removed. Outside this region, flank tissue (F) will form limb only when induced by
other tissues such as ear or nasal placodes. S, SS = scapula and suprascapula of the
shoulder girdle; C, PC = coracoid and procoracoid of the girdle; a, p, d, v =
anterior, posterior, dorsal and ventral poles of the principal transverse axes of the
limb; PN = pronephros; Si_12 = somites; Gi_3 = gills.
prospective free limb and girdle material. The remaining posterior half-ring of
limb disc tissue is actually peribrachial flank which does not normally participate in limb development. In our discussion of the literature, the term 'limb
region or disc' refers to this 3-5-somite-wide area.
The free limb area begins to thicken at stage 29, and becomes visible as a
growing bud by stage 36. The limb bud is conical at first and points in a posterior
-dorsal direction. At stage 40 the distal half of the bud flattens and undergoes
a torsion that orients the radial-ulnar plane approximately 30° to the vertical.
As the elbow joint and digits develop, the lower arm twists further to a vertical
alignment (Harrison, 1918).
It is commonly held that the limb rudiment is derived solely from the somatopleure of the lateral plate mesoderm in amphibians (Byrnes, 1898; Lewis, 1910;
Detwiler, 1918). However, a somitic contribution to the limb does not appear
to be ruled out. Recent studies on avian wing development, using marked somite
cells, have conclusively demonstrated that wing myogenic cells are derived
from the epibrachial somites (Dhouailly & Kieny, 1972; Chevallier, 1975,1978,
10
D. L. STOCUM AND J. F. FALLON
1979; Christ, Jacob & Jacob, 1977; Chevallier, Kieny & Mauger, 1977, 1978).
Further, it has been reported that somite cells are required for day-11 mouse
limb-bud development in organ culture (Agnish & Kochar, 1977). Circumstantial evidence for a somite contribution to the urodele limb comes from the
fact that the neurula-stage limb rudiment of T. pyrrhogaster will not develop
autonomously in vitro, but will develop if cultured with prospective somite
tissue (Amano, 1960). Furthermore, supernumerary limb buds can be evoked
in this species when ventral splanchnopleure is brought into contact with
somite tissue at the neurula stage. However, a clear interpretation of the role of
somite-derived cells in limb development is not possible from these experiments
because the somite or limb tissues were not marked in any way to determine
whether the somites actually contributed cells to the limb rudiments, or provided
an environment necessary for limb development. A somite contribution to the
limb rudiment is made more likely by the fact that limb buds develop from
masses of neurula stage A. maculatum somite plates wrapped in ventral ectoderm and cultured in vitro (Muchmore, 1957). In view of these results it would
be profitable to reinvestigate the question of a somitic contribution to the
limb regions in urodeles, using appropriately marked cells.
Regulative power of the limb region
Data from heterotopic transplantation experiments have suggested that the
forelimb of A. maculatum is determined as such by late gastrula (Detwiler,
1933). However, the frequency of autonomous differentiation of grafted limb
discs at this stage is very low, and Takaya (1941), on the basis of similar experiments with T. pyrrhogaster, found that determination of the forelimb mesoderm
may not be complete until the medullary fold stage, when the frequency of
self-determination rises considerably. The question of when the limb disc is
determined as limb is bound up with the question of whether its formation
requires a contribution from the epibrachial somite material. If there is such a
contribution, self-differentiation may not be possible until the neurula stage
because the disc does not contain a critical mass of prospective limb cells until
then.
Once determined, however, the limb region exhibits the classic morphogenetic
field property of regulation. Any of the four cardinal halves of the stage-29
A. maculatum limb disc can regulate to form a whole limb, although regulation
is observed more consistently with posterior and dorsal halves (Harrison, 1918).
It is noteworthy that the prospective shoulder girdle region is mosaic at this
stage (Detwiler, 1918). Hollinshead (1932) found that the dorsal half of the
A. maculatum limb disc grafted to the flank can regulate to form a normal limb
through stage 34, but limbs missing the radius developed with increasing
frequency between stages 35 and 37. However, dorsal and posterior halves
just of the free limb region of the disc developed into radially defective limbs
from stage 30 on, while anterior and ventral halves of the free limb region
Urodele limb ontogeny
11
regulated to produce normal limbs. These results indicate that, starting in the
posterior-dorsal quadrant, the prospective free limb cells begin to lose their
pleuripotency at stage 30 while the disc cells outside the free limb region retain
their potency longer.
A one-somite-wide ring of flank tissue surrounding the limb disc is also
competent to form a limb at stage 29 if the disc is completely removed (Harrison,
1918). According to Swett (1941), the replacement cells are derived mainly from
the dorsal sector of this tissue. This fact again raises the possibility that the
somites contribute some of the replacement cells. The remainder of the flank
tissue cannot regulate to form a limb after extirpation of the limb disc plus the
peribrachial ring of flank tissue (Harrison, 1918), but can be induced to form
limbs if stimulated by transplanted nasal or ear placodes in Bufo vulgaris
(Filatow, 1927), Triturus taeniatus, Hyla arborea, Rana esculenta (Balinsky,
1925; 1926, 1927a, b, 1933), Ambystoma maculatum (Glick, 1931) and Triturus
pyrrhogaster (Takaya, 1941; Ichikawa & Amano, 1949), and in A. maculatum
by implanting mouse kidney or transplantation of tail tissue (Holtfreter, 1955).
Balinsky (1933) obtained one case in which an extra limb was induced by
implantation of a piece of celloidin to the flank. Limbs were also induced in
A. mexicanum after grafting haploid forelimb buds to diploid flanks; the
induced limbs were diploid and arose after resorption of the transplants
(Hertwig, 1925, 1927). Thus, the flank mesoderm outside the immediate peribrachial region can be induced to express latent limb properties.
Limbs induced from the flank were of forelimb character when the inductor
tissues were implanted below the 4th to 7th somites, but were of hindlimb
character when the inductors were implanted below somites 9-14 (Balinsky,
1933). Either forelimbs or hindlimbs could be formed when the inductors were
placed under the 8th somite (Fig. 2). Furthermore, limbs were inducible all
along the flank, while pelvic girdles were inducible only under somites 11-13.
The frequency of limb formation declined steadily between somites 6 and 13.
Ectodermal-mesodermal interactions and limb outgrowth
The developmental specificity of the limb disc resides in its mesodermal component. Stage-29 forelimb ectoderm grafted to the flank does not form limb,
whereas stage-29 limb mesoderm grafted under flank ectoderm does so (Harrison, 1918) and experiments involving heteroplastic exchanges of prospective
forelimb ectoderm show that the chimaeric limbs develop with the size and
morphology of the host species (Rotmann, 1931). During larval stages, however,
the limb epidermis apparently does have a small, but noticable species-specific
effect on the limb morphology and size (Rotmann, 1933; Heath, 1953). Aside
from this effect, the ectoderm nevertheless has an indispensable role in limb
ontogeny and regeneration of all vertebrates. The apical ectoderm of the limb
bud of some anurans and all amniote embryos is thickened into a cap or ridge
(Saunders, 1948; Milaire, 1965; Tarin & Sturdee, 1971; Raynaud, 1972;
12
D. L. STOCUM AND J. F. FALLON
loor
90
80
70
60
50
40
30
20
10
8
9
10 11
Somite number
Forelimb induction
12
13
14
Hindlimb induction
Fig. 2. Frequency of induction of free limbs and pelvic girdles from flank tissue of
Triton taeniatus, by implantation of nasal placodes. The arrows beneath the abscissa
indicate the regions where forelimbs, hindlimbs, or either are induced. After
Balinsky (1933).
Stebler, 1973; Vasse, 1973; Fallon & Kelly, 1977). A similar ectodermal cap is
found on limb regeneration blastemas of larval and adult urodeles (Thornton,
1968), but not on their limb buds (Sturdee & Connock, 1975; Tank, Connelly &
Carlson, 1977). Regardless of its degree of thickening, removal of the apical
ectoderm or epidermis at progressively earlier stages of limb ontogeny or
regeneration results in progressively more truncated distal development (Steiner,
1928; Balinsky, 1935; Saunders, 1948; Goss, 1956; Tschumi, 1957; Amprino,
1965; Stocum & Dearlove, 1972). The commonly accepted role of the ectoderm
or epidermis in limb ontogeny or regeneration is one of maintaining subjacent
mesenchyme cells in an undifferentiated, dividing condition (Saunders, 1948;
Janners & Searls, 1971; Summerbell & Wolpert, 1972; Summerbell, Lewis &
Wolpert, 1973; Smith, Lewis, Crawley & Wolpert, 1974; Amprino, 1974;
Mescher, 1976). However, in addition to its postulated role in maintaining cells
in the cell cycle, the limb bud ectoderm or regenerate epidermis may also function to supply positional information specifying the distal boundary of the
limb, as will be elaborated below.
Determination of axial polarity
The classical test for the determination of limb axial polarity is to reverse one
of the three cardinal axes of the limb disc at different stages of development
Urodele limb ontogeny
13
while grafting the disc in either the orthotopic or heterotopic (flank) position.
If the limb develops with asymmetry opposite to that of its site of origin, the
polarity of the axis in question is not determined at the time of surgery. Using
this test, Harrison (1921) and Swett (1927, 1928«) concluded that the axes of
the urodele limb disc are determined in sequence. That the limbs developing in
these experiments were actually derived from the grafted discs was shown by
heteroplastic exchange between A. maculatum and A. tigrinum (Schwind, 1932).
By this test, the AP axis is already determined in A. maculatum and T. pyrrhogaster when the limb mesoderm becomes determined as limb; i.e. by the late
gastrula or medullary fold stages (Detwiler, 1929, 1933; Takaya, 1941). The
DV axis becomes determined between stages 33 and 35 in A. maculatum (Swett,
1927) and between stages 36 and 38 in A. tigrinum (Hollinshead, 1936) and
T. pyrrohgaster (Takaya, 1941). Although the embryonic stages at which the
DV axis is determined in A. maculatum and A. tigrinum are different, the gross
morphological stage of the limb disc is the same in both (Hollinshead, 1936).
The PD axis has been investigated only in A. maculatum, and becomes determined between stages 35 and 36, about the time the limb region begins outgrowth (Swett, 1927, 1928a). After reversal of the PD axis of a post-stage-37
limb bud, the bud tends to grow medially instead of laterally (Swett, 1927).
This direction of growth is blocked by the body wall, and the bud instead turns
and elongates in a posterior direction very close to the body wall. There is no
regeneration from the original proximal end of the limb, which often remains
as a visible swelling anterior to the growing tip. However, larval or adult forelimbs with reversed PD polarity can regenerate mirror image limbs from the
formerly proximal end of the reversed limb (Milojevic & Grbic, 1925; Dent,
1954; Butler, 1955; Deck and Riley, 1958; Wallace, 1980).
An important result of the axial determination experiments was the production of duplicated limbs after either orthotopic or heterotopic transplantations
(Harrison, 1921; Swett,1927). The primary limb developed first, maintaining the
AP polarity of the graft, while the secondary limbs began development somewhat later, and were mirror images of the primaries. The secondary limbs
produced after AP reversal in the orthotopic position arose either on the radial
side or ulnar side of the primary, or on both sides. The literature only infrequently contains a quantitative tabulation of the position of secondary limbs,
and our statements are based on descriptions given in the texts and figures of
Detwiler (1920), Harrison (1921, 1925), Swett (1926, 1927, 1930, 1932), and
Blount (1935). After AP reversal in the heterotopic position, the few secondary
limbs that developed arose on the radial side of the primary. When the AP axis
was not reversed in the heterotopic position, the secondary limbs again arose on
the radial side of the primary. These facts argue for some kind of interaction
between the limb disc and the adjacent flank tissue, the result of which is
different depending on the specific spatial relationships between the disc and
the surrounding flank tissue.
14
D. L. STOCUM AND J. F. FALLON
Table 1. Frequency of duplication (%) in the orthotopic or heterotopic positions
after reversal of the AP or DV axes or both, at different stages of ontogeny. A
more complete table may be found in the paper of Bryant & hen (1976)
No
Orthotopic
Stage 29-35a
A. mac.
Post-stage 35
1
A. mex.h
1
A. mac.0
1
N. virid°
Heterotopic
Stage 29-3 5a
A. mac.
Post-stage 35a
2
A. mac.
AP
DV
APDV
Rotation
85-7
6-3
747
0-0
72-9
87-5
1000
320
00
00
37-5
—
—
00
—
—
16-7
56-7
7-7
447
—
90-5
—
41-3
1
Axial reversals carried out on the tips of limb buds at stages 38-46.
Axial reversals carried out on whole limb discs or buds at stages 35-42.
a
Average values derived from the data of Detwiler (1920); Harrison (1921, 1925), Swett
(1926, 1927,1930,1932), and Blount (1935).
b
Average value derived from data of Maden & Goodwin (1980) and Thorns & Fallon (1980).
0
Data from Thorns & Fallon (1980).
2
Table 1 summarizes the frequency of duplication in A. maculatum and three
other urodele species after orthotopic or heterotopic transplantation, with or
without reversal of the AP, DV or AP and DV axes. The data allow four
generalizations: (1) The operation itself is not the cause of duplication. (2)
Duplication prior to stage 35 is most frequent in the orthotopic position after
reversal of the AP axis, regardless of whether the DV axis is simultaneously
reversed. (3) The frequency of duplication in the heterotopic position prior to
stage 35 is highest when thexAP axis is wo^versed, regardless of whether the
DV axis is simultaneously reversed. (4) There rs^a high frequency of duplication
in post-stage-35 A. maculatum and A. mexicanum limb buds after reversal of
either their AP or DV axes. Duplication also occurs at a high frequency after
either AP or DV orthotopic axial reversal of regeneration blastemas (Milojevic, 1924; Iten & Bryant, 1975; Tank, 1978; Maden & Turner, 1978). When
the AP axis is reversed, the secondary limbs have the same relationships to their
primaries as are observed after AP reversal of limb discs in the orthotopic
position. These results suggest that prior to stage 35, duplication occurs only
after changing the spatial relationships of the AP axis of the limb discs with
respect to the surrounding tissues. A comparable change of the DV axis alone
elicits practically no duplication. After stage 35, however, reversal of the DV
Urodele limb ontogeny
15
axis alone also results in the production of supernumerary limbs. The fact that
supernumerary limbs can be produced by AP reversal is consistent with the
conclusion that the AP axis is determined before the DV axis, if it is assumed
that after its determination, the DV axis interacts with surrounding flank tissues
differently than it does prior to its determination. In the latter case, the interaction leads to repolarization; in the former case, it leads to supernumerary
production (as in the case with the AP axis).
The formation of supernumerary limbs after axial reversal of regeneration
blastemas or post-stage-35 limb bud tips can be accounted for by models which
postulate that, when confronted, the normally 180° opposite cells of host and
graft reciprocally interact to stimulate mitosis and fill in, by intercalary regeneration between the poles, all the positional values specifying pattern in the
transverse plane (Carlson, 1975; Bryant & Iten, 1976; French et al. 1976;
Bryant & Baca, 1978; Stocum, 1980fl). Once these supernumerary values are
rilled in, distal transformation can create a supernumerary limb. The supernumerary limbs are usually derived from both graft and host cells (Tank, 1978;
Holder & Tank, 1979; Stocum, 1980a, b). However, the ability of these models
to predict the number and especially the location of supernumeraries has been
challenged in studies on regenerating urodele limbs (Maden & Turner, 1978;
Wallace & Watson, 1979).
An intercalary regeneration model has also been applied to explain the
duplications that occur after grafting embryonic limb discs (Bryant & Iten,
1976). The duplications that occur on the radial side of the primary after AP
reversal of the disc in orthotopic position would be due to intercalary regeneration between the anterior edge of the limb disc and the peribrachial flank tissue
posterior to it. Duplications forming on the ulnar side would result from intercalary regeneration between the posterior edge of the limb disc and the peribrachial flank tissue anterior to it. The duplications observed on the radial side
of the primary after grafting discs to the flank without AP reversal would be the
result of intercalary regeneration between the anterior edge of the limb disc
with the posterior edge of the flank tissue that is capable of forming forelimb
when stimulated by inductors. However, intercalary regeneration models
predict no duplications after grafting a limb disc to the flank with reversed
AP axiation, a situation where the anterior edge of the forelimb disc would
supposedly contact the anterior edge of the hindlimb field. In reality, this
operation results in a 16-7% incidence of duplication after reversal of just the
AP axis, and a 7-7 % incidence of duplication when both AP and DV axes are
reversed (Table 1). Finally, intercalary regeneration models of supernumerary
limb formation depend upon the assumption that the AP polarity of the
embryonic limb rudiment is determined prior to stage 35, a notion we argue
against after presenting an alternative model.
16
D. L. STOCUM AND J. F. FALLON
G2 % G 3
Fig. 3. Polarizing model for determination of the A? and DV axes. The classical
limb disc, containing limb tissue (FL), shoulder girdle (SG), and posterior peribrachial flank tissue (PBF) is outlined by the large circle. The X's indicate tissue
inferred to have a DV polarizing effect, the dots indicate tissue inferred to have an
AP polarizing effect. The arrows show the direction of spread of the polarizing
signals across the limb mesoderm in the normal embryo. The signals can spread
in any direction from the polarizing tissue as shown by experiments in which
dorsal polarizing tissue is rotated with respect to the limb tissue (Swett, 1938).
PN = pronephros; a, p, d, v = poles of the AP and DV axes; Gi_3 = gills.
The polarizing model
We propose a polarizing model that can account for both the genesis of
axial polarity during limb ontogeny and the known facts about limb duplication
after orthotopic or heterotopic transplantation of limb discs. In this model,
posterior and dorsal peribrachial flank mesoderm specify AP and DV polarities
in the adjacent limb mesoderm by means of signals which spread across the
prospective limb mesoderm (Fig. 3). The polarizing tissues need not participate
in the development of the limb, nor be reciprocally influenced by the limb tissues,
but these possibilities are not ruled out. The AP polarizer is the posterior tissue
of the classical limb disc that does not become part of the limb or girdle, and
the DV polarizer is the flank tissue in the region of the pronephros. We now
develop the argument for this model.
The importance of peribrachial flank tissue to forelimb development has been
shown by several studies. Stage-29 limb discs transplanted to the dorsal or
ventral midline (Nicholas, 1924a; Takaya, 1941; Finnegan, 1960), to the side
of the head between eye and gill (Slack, 1977) or cultured in vitro (Wilde, 1950)
fail to develop unless peribrachial flank tissue is included. That this developmental failure is not due to nonspecific effects related to the suitability of the
transplantation site is shown by the fact that a limb disc grafted first to the
head, then regrafted to the flank with a ring of head tissue around it also fails to
develop (Swett, 1945). The postural orientation of the limb corresponds to the
orientation of the ring of peribrachial flank tissue, not to the body of the embryo
Urodele limb ontogeny
17
as a whole, and duplication occurs only when the limb disc is disharmonic to the
peribrachial ring, not when the ring plus limb tissue is disharmonic to the body
(Nicholas, 19246, 1958; Swett, 1945). Furthermore, duplication effects are
not associated with tissues other than flank tissues (Detwiler, 1930; Swett,
1945).
The idea that the developmental importance of flank tissue is due to an axial
polarizing activity was first raised in connection with determination of the DV
axis of the urodele forelimb. Swett (1938) found that if dorsal peribrachial
flank tissue alone was included with a stage-29 A. maculatum limb disc grafted
to the flank with its DV axis reversed, repolarization of this axis to harmonize
with the DV axis of the body was prevented. When the same experiment was
made including ventral, instead of dorsal peribrachial tissue, the DV axis was
repolarized. However, dorsal peribrachial tissue transplanted ventral to a flank
grafted limb disc having harmonic DV orientation was unable to reverse the
DV polarity of the disc. These results led Swett (1938) to view the dorsal peribrachial tissue in these experiments as a physical barrier to other DV polarizing
factors in the flank. From his discussion, one would conclude that these factors
are ventrally located. The interpretation of Swett's results is complicated,
however, by the fact that the transplantations were made to the flank, the
dorsal portion of which he found to have effects similar to those of dorsal
peribrachial tissue, thus failing to isolate the dorsal peribrachial tissue as the
sole variable in the experiments. Takaya (1941) transplanted limb discs with
attached dorsal or ventral peribrachial tissue to the ventral midline (where
limb discs alone fail to develop), and found that the discs developed only when
the dorsal tissue was included. This result suggests that the dorsal peribrachial
tissue is indeed acting as a polarizer, specifying the dorsal-ventral axis, but
these ventral midline experiments need to be repeated placing dorsal peribranchial tissue on the ventral side of the disc to see if this operation will reverse
the DV polarity of the limb.
A zone of posterior mesodermal tissue which polarizes the AP axis has been
postulated for the limb buds of Xenopus (Cameron & Fallon, 1977), reptiles,
birds and mammals (Fallon & Crosby, 1977). The major evidence for this zone
is that in chick limb buds posterior border tissue grafted to the anterior border
stimulates the production of mirror-image supernumerary limb structures, with
the most posterior part of the supernumerary arising adjacent to the graft
(Saunders & Gasseling, 1968; MacCabe, Gasseling & Saunders, 1973; Fallon &
Crosby, 1977; Summerbell & Tickle, 1977). Therefore, this region along the
posterior limb bud has been called the zone of polarizing activity or polarizing
zone (Balcuns, Gasseling & Saunders, 1970). It is clear that the ectoderm of the
posterior border need not be present and that the orientation of the graft
itself does not change the result (Fallon & Thorns, 1979). Further, the graft
need not take part in the supernumerary outgrowth (Smith, 1979, 1980; Fallon
& Thorns, 1979). The wing supernumerary limb elements that do form can be
18
D. L. STOCUM AND J. F. FALLON
altered by irradiating quail polarizing zone in increasing doses between 10000
and 90000 rads before grafting to the chick wing anterior border. At low doses,
supernumerary digits 4,3 and 2 form, at intermediate doses only supernumerary
digits 3 and 2 form, and at high doses, only a supernumerary digit 2 forms.
With the highest doses, no supernumerary limb elements form at all (Smith,
Tickle & Wolpert, 1978). The quail polarizing zone does not contribute cells to
the supernumeraries formed in these experiments. These results suggest the
possibility that at increasing dose levels fewer surviving cells were left to contribute whatever stimulus comes from polarizing zone.
Since the polarizing zone was discovered, there has been controversy about
its role in normal limb development. One major theory is that polarizing zone
is the source of a diffusible morphogen. It is proposed that the morphogen is in
a gradient across the limb bud such that the highest concentration is posterior,
intermediate concentrations are in the middle, and the lowest concentration is in
the anterior bud. This would bring about specification of polarity along the
anteroposterior axis. Thus, grafting a second polarizing zone would set up a
second morphogen gradient resulting in supernumerary outgrowth (reviewed
in Summerbell, 1979; Tickle, 1980a). There are various lines of evidence
supporting this point of view. However, possibly the most convincing data come
from the work of Summerbell (1979) who showed that an impermeable barrier
placed through the limb bud (ectoderm and mesoderm) at right angles to the
body wall caused deletions anterior to the barrier. He interpreted this to mean
that the morphogen from polarizing zone was blocked by the barrier and this
caused the deletions.
The fact that the apical ridge was also blocked, i.e. anterior ridge was separated
from posterior ridge, led to the suggestion that this separation in itself might
cause the observed anterior deletions. It was demonstrated that surgical removal of posterior ridge in the wing bud did result in the failure of anterior
structures to develop even though anterior ridge remained. These results were
similar to those in SummerbelPs barrier experiment. Furthermore, there seemed
to be a critical portion of posterior ridge which, once removed, resulted in the
failure of expected anterior structures (Rowe & Fallon, 1981). Equally important is the fact that impermeable barriers grafted to the chick leg bud do not
result in deletions anterior to the barrier (Rowe & Fallon, 1982), nor for that
matter does posterior apical ridge removal in the leg result in anterior deletions
(Rowe & Fallon, 1981). One is left with the conclusion that either the barrier
does not block the proposed morphogen as Summerbell (1979) supposed or
that morphogen is not needed for polarization along the AP axis during the
developmental times studied. We stress that, at present, there are no unequivocal
data showing that polarizing zone has any role in polarizing the mesoderm
during the limb bud stages of development. This is consistent with the facts that
the wing bud develops normally following removal of the polarizing zone
(MacCabe, Gasseling & Saunders, 1973) and the zone does not regenerate after
Urodele limb ontogeny
19
simple surgical removal (Fallon & Crosby, 1975; MacCabe, Knouse & Richardson, 1981).
It has been postulated that polarizing zone is not important for limb development at all. Saunders (1977) proposed this because he found polarizing activity
in other than limb tissues, lten and co-workers (Iten & Murphy, 1980; Javois &
Iten, 1981; Javois, Iten & Murphy, 1981) claim no special significance for the
polarizing zone on the basis of a set of experiments where nonadjacent wedges
of limb mesoderm and overlying ectoderm were grafted into slits made into
the host limb bud. Supernumerary structures formed even though the graft
was of an anterior wedge to an anterior site. She interpreted these and other
experiments to mean that the disparity in positional values caused intercalary
replacement of the missing positional values and resulted in a supernumerary
outgrowth. In this context, Iten sees no special properties associated with the
polarizing zone.
There is a question whether Iten's data relate to the issue of whether or not
there is a polarizing zone and whether it has a role in limb development. First,
polarizing zone mesoderm without ectoderm will cause duplications. Iten
always grafts the apical ectoderm with the wedges of mesoderm. Second, if
anterior wedges of quail limb bud tissues are grafted to posterior sites in chick
limb buds, in the manner described by Iten and co-workers, the supernumerary
structures formed are composed entirely or almost entirely of quail cells
(L. Honig, personal communication). This result differs from that obtained
after grafting polarizing zone anteriorly. Here, the polarizing zone graft need
not take part in the supernumerary, nor in fact need it be present after 12-15 h
in place (Smith, 1979, 1980; Fallon & Thorns, 1979 and unpublished). Furthermore, if quail limb buds are irradiated in a dose that permits polarizing zone to
act (see above) and anterior wedge-shaped pieces cut from them and grafted to
posterior locations in unirradiated chick limb buds, no supernumerary structures
develop (L. Honig, personal communication). This is different from other
systems (Drosophila imaginal discs, regenerating amphibian limbs) in which
positional discontinuity between irradiated and unirradiated tissue evokes
supernumerary formation from the unirradiated component (Adler & Bryant,
1977; Maden, 1979). Third, as few as 100 polarizing zone cells, disassociated
and grafted to the anterior wing border will evoke supernumeraries (Tickle,
19806). A comparable experiment using as few displaced anterior cells grafted
in the same way (Tickle, 19806) should result in supernumerary outgrowth if
Iten is correct. Fourth, polarizing zone will inhibit recombinant limb development (MacCabe, Saunders & Pickett, 1973; Crosby & Fallon, 1975; Frederick
& Fallon, 1982). No other limb or flank cells have this property. Finally, Honig
(1981) has shown that when polarizing zone tissue and a block of anterior leg
tissue are grafted in tandem to the anterior border of the chick wing (with the
leg graft posterior to the polarizing zone graft) the host anterior wing tissue
next to the leg tissue produces mirror-image supernumerary structures. Anterior
20
D. L. STOCUM AND J. F. FALLON
lOOi—
90
80
70
60
K
50
40
a.
a
30
20
10
0
Somite number
Fig. 4. Frequency of duplication when progressively more posterior flank tissue
is placed contiguous to anterior limb rudiment tissue, by (a) autoplastic grafting of
the limb disc to progressively more posterior locations on the flank (O—O; A.
maculatum) (Detwiler, 1920); {b) homoplastic grafting of the limb disc in the
same fashion ( # — # ; A. maculatum) (Detwiler, 1920); or (c) heterografting
progressively more posterior strips of flank tissue (one somite wide) adjacent
to the anterior border of the limb rudiment ( A — A ; A. mexicanum) (Slack,
1976). In the A. maculatum experiments, the limb rudiment was positioned so that
its anterior border was at the flank levels indicated by the somite numbers. Note
that the duplication frequency is highest from the 5th to the 8th somites, decreasing
sharply under the 4th and 9th somites.
leg tissue grafted alone does not result in supernumerary formation. The leg
tissue has been grafted with and without apical ridge (L. Honig, personal
communication). This result signifies that the posterior border tissue acts on
the host wing tissue at a distance, not by intercalation initiated by local interactions between the edges of a positional discontinuity, and constitutes strong
evidence for a posterior polarizing region. All of these data suggest it is the
graft that grows out in Iten's experiments and this is quite different from the
outgrowths caused by polarizing zone, where anterior tissue grows out. The
fact remains that there is a demonstrable high point of morphogenetic activity
along the mesoderm of the posterior border of the chick and all other amniote
limb buds tested to date.
Urodele limb ontogeny
21
Since it appears that posterior border tissue is not required for AP polarization
in the growing chick limb bud, Fallon & Crosby (1977) have suggested that a
polarizing region of flank tissue determines the AP axis of the prospective chick
limb mesoderm prior to its outgrowth as a bud, and is not thereafter required
for normal limb development, although its continued (but residual) activity
can be demonstrated by grafting. Fallon & Crosby's (1977) notion has not been
tested on early chick embryos, but is consistent with the fact that the transverse
axes of the prospective urodele forelimb are polarized prior to outgrowth of the
limb as a bud, and receives support from other studies on urodele limb ontogeny.
First, Swett (1926, 19286) showed that when the limb disc of a stage-27 to -34
A. maculatum embryo was separated into anterior and posterior halves by a
thin strip of flank tissue, two primary limbs with the same asymmetry were
formed. However, a mirror-image secondary limb usually arose on the radial
side of the more posterior primary member, suggesting a selective repolarization
of a portion of this member by the grafted flank strip. Second, the posterior
edge of the limb disc (the portion which does not participate in the formation
of the limb), as well as flank tissues from the 5th to the 8th somites, can stimulate the production of a mirror-image secondary limb on the radial side of a
limb disc when in contact with that side (Detwiler, 1920; Slack, 1976). The
mesoderm, not the ectoderm, is the effective component. Taken as a whole, the
data indicate that the ability of flank tissues to effect duplication is high from
somites 5 (non-limb tissue that is part of the classical limb disc) to 8, exhibiting
an increase between these two points, and declining sharply under somites 4 and
9 (Fig. 4). Anterior limb disc or anterior peribrachial tissue grafted to the
posterior edge of the limb does not result in duplication (Takaya, 1941; Slack,
1976).
The concept of an AP polarizing effect, in which a large region of flank tissue
can specify adjacent limb-forming mesoderm as most posterior, can explain the
frequency, location, and handedness of duplicated limbs arising after orthotopic
and heterotopic transplantations of urodele limb discs (Fig. 5). As seen from the
map of the forelimb region (Fig. 1), the transplanted limb discs of most investigations have contained polarizing flank tissue on their posterior edge. The high
frequency of duplication on both the radial and ulnar sides of primary limb
discs grafted in the AP-reversed orthotopic position would be due to the fact
that polarizing tissue is put in contact at both these loci with anterior tissue
having limb-forming capability. The high frequency of duplication on the
radial side of limb discs grafted to the flank without AP reversal would be the
result of placing the anterior edge of the limb disc within a flank region of
high polarizing activity. No duplications would arise on the ulnar side of these
grafts because the polarizing tissue of their posterior edge does not induce
duplications from flank tissue. Duplications would be expected to arise at the
observed low frequency on the radial side of AP-reversed limb discs grafted to
the flank because the anterior edge of the disc would sometimes lie within the
22
D. L. STOCUM AND J. F. FALLON
(A) Orthotopic:
axial reversal
(B) Heterotopic:
no axial reversal
(C) Heterotopic:
axial reversal
Urodele limb ontogeny
23
rapidly falling gradient of polarizing activity between the 8th and 9th somites,
but would lie completely outside the polarizing region in the vast majority of
grafts. Similar conclusions based on an AP polarizing action of flank tissue
have been drawn by Slack (1976, 1977).
The polarizing model predicts that the secondary limbs produced after
grafting limb discs to the flank should all arise from the graft. This prediction
has been borne out in experiments by Swett (1932) in which limb discs were
heteroplastically exchanged between A. maculatuma. nd A. tign'num, although
Scharrer (1931), on the basis of similar heterografting experiments using the
same species, has claimed that some of these limbs can arise partly from host
flank tissue. The model also predicts that secondary limbs produced on the
ulnar side of limb discs grafted in the AP-reversed orthotopic position should
arise from respondent anterior peribrachial tissue, while those produced on the
radial side should arise from respondent anterior limb disc tissue. Although
Harrison (1921), Swett (1926), and Detwiler (1930) state, on the basis of external
morphology, that secondary limbs always originate from the grafted disc, no
cell or tissue markers were employed in their orthotopic transplantations.
Scharrer (1931) showed by heteroplastic grafting that secondary limbs produced
in the orthotopic position can arise entirely from host or graft tissue, which is
in general accord with the prediction. However, he did not make any specific
correlation between the tissue origin of the secondary limbs and their radial or
ulnar location, and this type of experiment should be repeated with such a
correlation in mind. Nevertheless, Slack (1976) has shown that secondary limbs
formed after heterografting flank tissue adjacent to the anterior edge of the
limb disc in orthotopic position are always derived from the limb disc tissue,
as predicted by the polarizing model.
Fig. 5. Diagrams indicating how the polarizing model accounts for the location and
handedness of duplications after orthotopic and heterotopic transplantation of limb
discs at stage-29. The upper diagrams of each set depict the grafting operation and
the tissues from which the primary (1°) and secondary (2°) limbs are predicted to
arise. The arrows show the origin, direction, and extent of penetration of the
anterior-posterior polarizing effects of the flank tissue on the posterior side of the
disc and the remaining flank tissue surrounding the disc. The dorsal-ventral axis
is repolarized to normal at stage-29 whenever it is reversed by the operation (Swett,
1927), and is pictured this way in the diagrams. The lower diagrams of each set show
the symmetry relations of the fully developed primary and secondary limbs, a, p,
d, v = poles of the anterior-posterior and dorsal-ventral axes; L, R = left and
right. (A) Orthotopic transplant of either a right limb disc to the right side, reversing
the AP and DV axes, or a left limb disc to the right side, reversing only the
AP axis. (B) Heterotopic transplant of right limb disc to the right side, no axial
reversal, or a left limb disc to the right side, reversing only the DV axis. (C)
Heterotopic transplant of either a right limb disc to the right side, with AP and DV
axial reversal, or a left limb disc to the right side, reversing only the AP axis. The
frequency of duplication after the latter transplantations is much lower than after
any other, because the anterior edge of the grafted disc is in a flank region having
little or no polarizing activity.
24
D. L. STOCUM AND J. F. FALLON
The polarizing model also offers a possible explanation for a puzzling aspect
of the development of limbs induced from flank tissue by nasal placodes and
other inducers. These limbs were usually of reversed AP polarity and normal
DV polarity (Balinsky, 1933; Ichikawa & Amano, 1949). Furthermore, Takaya
(1941) showed that limbs of normal AP and DV polarity were formed after
grafting inducers into blocks of flank tissue whose AP axis or AP and DV axes
were reversed. Takaya's flank tissue blocks were 3-5 somites in width and always
included tissue posterior to the 8th somite. These facts suggest that the disharmonic AP polarity of limbs induced without flank axial reversal is associated
with the AP asymmetry of the steep gradient in polarizing activity posterior to the
8th somite. This asymmetry is reversed in the flank tissue rotation experiments,
so that it has the same polarity as the gradient from the 5th to the 3rd somites.
Takaya's reversed grafts did not include dorsal flank polarizing tissue; thus the
DV axis of the induced limbs would have been repolarized to normal. This
hypothesis suggests that limbs induced between somites 5 and 8, where AP
polarizing activity is high, would develop as symmetrical double posterior
limbs. Slack (1977) has noted that a number of the flank-induced limbs illustrated in the literature appear to be symmetrical in their AP axis.
The nature of the polarizing signal is unknown. The positional information
of the polarizing zones could be transmitted across the prospective limb mesoderm in the form of a gradient of diffusible morphogen, with each concentration
specifying a positional value to be interpreted by the cell and transduced into
the proper pattern of gene activity (Tickle, Summerbell & Wolpert, 1975).
The morphogen could diffuse through the extracellular spaces from its source
or from cell to cell via gap junctions (Furshpan & Potter, 1968; Wolpert,
1978; Bennett, Spray & Harris, 1981). Alternatively, the polarizing effect could
be in the form of a gradient of cell surface molecular organizations, with the
polarizing zones acting as origins of the gradient, which could be developed by
local interactions starting at the junction of the zone and the adjacent prospective
limb tissue and proceeding in sequence across the prospective limb tissue.
Likewise, the interactions between DV and AP polarizing activities are unknown.
The same kind of molecules and mechanisms may be involved in establishing
both polarities, or the molecules and/or mechanisms may be different in each
case.
Simultaneous determination of AP and DV axes
The polarizing model raises the possibility that the determination of the AP
axis before the DV axis in the prospective forelimb is an illusion. The error
would be created by the fact that in the flank grafting experiments used to test
this determination, the grafted tissue contains AP polarizing mesoderm and/or
is embedded in it. Thus, a stage-29 limb disc with an undetermined AP axis
would behave in reversal experiments as if this axis were determined, due to the
action of its polarizing tissue, just as the DV axis behaves as if determined when
Urodele limb ontogeny
25
reversed with attached dorsal peribrachial tissue (cf. Swett, 1938; Slack, 1977).
An interesting aspect of radially duplicating limbs is indicated by one of
Swett's (1940) experiments. He found that the number of mitoses observed in
the early stages of a radially duplicating limb bud was no greater than the
number observed in the contralateral, non-duplicating bud. This observation
suggests that the cells of the anterior half of the original limb rudiment are
being reassigned AP positional values in a mirror-image sequence by adjacent
polarizing tissue. Cells of the posterior half of the original rudiment would also
have their positional values reassigned to restore a complete set of AP values
of the original asymmetry. Slack (1980) has also stated that half-limb rudiments
which regulate to form whole limbs do not show a larger number of mitoses
next to the cut edge. The significance of these observations, however, requires
further evaluation, for neither Swett nor Slack presented data on the total
number of cells, i.e. a mitotic index was not calculated in the experimental and
control buds.
Evidence for the plasticity of limb AP polarity throughout tailbud stages
comes from studies on the hindlimb region. Stultz (1935) grafted AP-reversed
stage-35 A. maculatum hindlimb areas homoplastically or heteroplastically (to
A. tigrinum) to the flank, or in orthotopic position (the development of the
hindlimb is 2-3 weeks behind that of the forelimb). In the heterotopic
position, the grafted rudiments developed with reversed AP polarity, but
in the orthotopic position, they developed with normal AP polarity. These
results suggest that at this stage, the AP axial polarity of the hindlimb
rudiment can easily be reversed by the tissues immediately surrounding the
orthotopic site.
However, the most intriguing evidence for labile AP polarity during tailbud
stages, and its determination by polarizing flank tissue, involves a relationship
between limb and gill tissues. Detwiler (1922) found that limb discs transplanted
in place of gill tissue developed poorly. Wilde (1952 a) subsequently carried out a
series of ingenious experiments on stage-28 to -38^4. maculatum embryos, in
which the positions of gills 1-2 and the limb disc were reversed by 180° rotation
of the limb-gill complex (leaving gill 3 in place), thereby placing gill tissue anterior
and posterior to the limb disc. The operations were done in such a way that the
AP and DV axes of both limb and gill were either reversed or remained harmonic with respect to the body. From stages 28-33, when the AP axis is supposedly determined and the DV axis is not, limb development was inhibited.
Normal limbs developed from transplants done at stages 35-38. Wilde (19526)
also conducted in vitro studies which demonstrated a similar time course for the
acquisition of self-organizational capacity by the limb disc or bud in the presence
of gill tissue. The suppressive effect on self-organization was proportional to
the amount of gill tissue in the explants, and the limbs exhibited radial symmetry of what little differentiation there was, instead of the expected asymmetry.
Wilde (1952#) noted that the time at which the limb rudiment acquired the
26
D. L. STOCUM AND J. F. FALLON
ability to develop in the presence of gill tissue coincided with determination
of the DV axis. This fact suggests that outgrowth and morphogenesis of the
limb cannot begin until the DV axis has become polarized. We might then
conclude that the failure of pre-stage-35 limb discs to develop in these experiments is due to inhibition of DV axial determination by gill tissue, even though
the AP axis is already determined. However, Wilde's diagrams show that
dorsal peribrachial (polarizing) tissue was included in his grafts, and that this
tissue was not in contact with gill tissue. Therefore, the DV polarizing region
of pre-stage-35 limb discs should be present in these experiments. We are then
led to the conclusion that development of these discs fails because their AP
polarity has not yet been established and is prevented by adjacent gill tissue
from becoming established. The effect of the gill tissue would be to specifically
inhibit the AP polarizing activity of the flank mesoderm in the posterior part
of the limb disc. Specific inhibition of posterior disc tissue is indicated by the
fact that transplantation of gill tissue to the anterior edge of the hindlimb rudiment does not inhibit hindlimb development (Wilde, \952a).
The foregoing discussion not only leads to the conclusion that neither the
AP nor the DV axes of the prospective limb tissue are determined prior to
stage 35, but that both axes must be determined before limb outgrowth and
morphogenesis can begin. This conclusion is also supported by the fact that
limb discs (which contain AP polarizing tissue) grafted to the ventral midline
with attached dorsal peribrachial tissue alone will produce limbs. However, if
limb discs are grafted with attached ventral peribrachial tissue alone, they fail
to develop (Takaya, 1941). Recently, Slack (1980) has made the very important
rinding that half or double half limb rudiments will not develop when grafted
to the head unless both posterior and dorsal peribrachial flank tissue are present
in the graft. In view of these facts, we suggest that the following two concepts
are central to the understanding of limb ontogeny: (1) The AP and DV axes
of the prospective vertebrate limb mesoderm are determined together, and
(2) determination of both these axes is a prerequisite for the outgrowth and
polarization of the PD axis.
Proximal-distal outgrowth
Since amphibian limb parts are laid down in a proximodistal sequence
under the influence of the apical ectoderm (Tschumi, 1957), the dividing cells
of the limb mesoderm must change positional value in a distal direction as the
disc grows out into a bud. The growing limb bud is a three-dimensional structure
with internal pattern, therefore its parts must be specified by at least three
positional coordinates (values). Two of these values specify position in the
transverse plane, while the third specifies position on the PD axis. Thus, each
cell can detect its position relative to each of its neighbours, and the prospective
tissue pattern can be represented as a map of positional values. The transverse
positional values are specified by the action of the dorsal and posterior polariz-
Urodele limb ontogeny
Fig. 6. Boundary model of proximal-distal limb bud outgrowth. (A) Transverse
section through a right limb rudiment (L) whose AP and DV axes have just been
determined, but which has not yet begun outgrowth. The mesoderm cells of the rudiment in contact with the overlying ectoderm (E) assume the most distal (Dsj positional value as a result of this contact. The remaining cells have the most proximal
(Pr) positional value. These boundaries (stippled cells) represent the proximal-distal
axial outline. The discontinuity will be filled in by cell division and continual distal
averaging of positional values by the daughter cells (unstippled) (Maden, 1977). (B)
Three-dimensional cutaway diagram of a growing undifferentiated right limb bud,
showing the boundary shell of mesoderm cells in transverse and longitudinal section.
The arrows indicate the distal direction of positional value change, a, p, d, v = poles
of anterior-posterior and dorsal-ventral axes.
27
28
D. L. STOCUM AND J. F. FALLON
ing regions, and once this specification has taken place, the limb rudiment can
grow out and the PD values can be specified. The morphogenetic field of the
limb may thus be viewed as a set of AP, DV and PD boundary positional
values which prescribe the structure to be developed (Fig. 6). The AP and DV
boundary values will be those represented by the cells at the axial poles of the
transverse plane. The ability of the limb area to develop autonomously prior
to stage 35 thus requires that the polarizing tissues be included with the prospective limb tissue, whereas self-organization can proceed in their absence after
this stage.
The proximal boundary value is that value intrinsic to the limb mesodermal
cells prior to the initiation of limb outgrowth, and it specifies the base of the
limb. We now make the important provision that cells of the layer of limb
mesoderm in contact with the overlying ectoderm must assume the most distal
boundary value as a result of that contact (Maden, 1977;Stocum, 1978, 1980o).
Further, the dividing cells between the two boundaries or their progeny are
free to change their positional values in a distal direction. The PD positional
value map isfilledin by intercalation between the boundaries, possibly involving
a synchronous averaging mechanism (Maden, 1977). All the mesodermal cells
are continually monitoring the positional values of their neighbours during limb
ontogeny, and are consequently acting as local boundaries. However, the most
proximal and most distal boundaries are special in that all changes in positional
value are ultimately referenced to them.
We postulate as others do (Maden, 1977) that positional value may be reflected in the molecular organization of the cell surface coat. Thus, in establishing
the PD limb positional value map by intercalation, the mesoderm cells interact
in an integrated network of strictly local communications. Since the structural
details of the limb in the transverse plane are different at different levels of the
PD axis, changes in AP and DV positional values during limb outgrowth must
be coupled to changes in transverse values. We postulate an intracellular integrating mechanism in which changes in PD value simultaneously effect changes
in transverse values so that they correspond to the new PD level. The three
cardinal axes of asymmetry thus develop interdependently during limb outgrowth (see also Thorns & Fallon, 1980).
Relationship of limb ontogeny to regeneration
The limb regeneration blastema is formed by the dedifferentiation of stump
tissues, and it redifferentiates a replica of the parts lost by amputation. The
blastema is a polarized, self-organizing structure that undergoes autonomous
^differentiation and morphogenesis when isolated from its stump (Stocum,
1968, 1978, 19806; de Both, 1970; Michael & Faber, 1971; Dinsmore, 1974).
The morphogenetic field of the regeneration blastema can thus be considered as
a set of boundaries of the structure that is to be regenerated, equivalent to that
of the limb bud, except that the proximal boundary of the regenerate is deter-
Urodele limb ontogeny
29
mined by the positional value of the cells at the level of amputation, and the
distal boundary is imposed by the apical wound epidermis (Maden, 1977;
Stocum, 1978, 1980a). The supernumerary limbs that form at the graft-host
junction after reversing the AP or DV axis of the blastema with respect to the
stump can be attributed to the intercalation of new sets of transverse positional
values, using the apposed axial poles represented by blastema and stump as
boundaries, followed by intercalation of PD values. Likewise, the boundary
model explains why tissues internal to the skin (muscle, bone) can be removed
from the limb, yet be regenerated distal (but not proximal) to the amputation
plane by the blastema derived from the remaining tissue (Bischler & Guyenot,
1925; Weiss, 1926; Goss, 1957; Carlson, 1972). After these operations, either
muscle, skin or both are retained in a normal configuration. These tissues
contain the positional values representing the entire periphery of the transverse
plane of the limb. Therefore, when the cells of these tissues dedifferentiate,
opposite positional values will be brought into contact, and intercalation of
those values internal to the periphery will ensue.
A major question is whether specification of the map of transverse positional
values in the regeneration blastema, or the production of supernumerary
regenerates, requires the renewed activity of latent polarizing zones incorporated into the limb during ontogeny. This possibility is unlikely because exclusion
of posterior tissue (from which the AP-polarizing zone would be activated) from
a limb does not render it incapable of regeneration. Thus, double anterior-half
wrist blastemas evoke intercalary regeneration from double anterior-half
thighs when grafted to them (Stocum, 1981). The production of both primary
and supernumerary regenerates can best be explained as a result of reciprocal
stimulation and intercalary regeneration between boundary cells whose positional values were established during ontogeny, and inherited by blastema cells
during dedifferentiation, or imposed by contact with apical wound epidermis
(distal boundary, Maden, 1977).
CONCLUSIONS
The results of numerous experiments described in the literature of the past
70-80 years suggest the following concepts of urodele limb ontogeny: (1) The
AP and DV axes of the limb rudiment are not determined in sequence, but
simultaneously between stages 33 and 35, by the action of dorsal and posterior
polarizing tissues, and (2) determination of the PD axis and outgrowth of the
limb bud cannot begin until the AP and DV axes of the limb rudiment are
determined. We emphasize that these assertions are by no means proven;
however, their antecedent arguments suggest a variety of experiments to test
them.
The morphogenetic field of the limb rudiment is initially unpolarized, or
has only a very weak polarity. Dorsal and posterior regions of peribrachial
2
EMB 69
30
D. L. STOCUM AND J. F. FALLON
flank tissue polarize the AP and DV axes of the field, assigning to its cells a
set of positional values specifying pattern in the transverse plane. Proximaldistal outgrowth then takes place, generating, between the distal and proximal
boundaries of the field, the set of positional values that specifies the PD axis of
the limb.
During the regeneration which occurs after amputation of a part of the
urodele limb, the distal boundary of the limb must be reconstructed, and is
imposed upon the apical layer of blastema cells specifically by the apical wound
epidermis. The proximal boundary of the regenerate is the PD value, recorded
during ontogeny, of the PD level of origin of those blastema cells not in contact
with the apical wound epidermis. As in limb bud outgrowth, a complete set of
PD positional values is restored by distal transformation of blastema cells,
using the boundaries as reference points.
We thank the following people for helpful discussion and criticism: Susan Bryant, Bruce
Carlson, Charles Dinsmore, Richard Goss, Malcolm Maden, Paul Maderson, James Nardi,
Donene Rowe, B. Kay Simandl, David Slautterback, Patrick Tank, Stephen Thorns and
Charles Wilde. We are indebted to Lawrence Honig for permitting us to cite some of his
work in advance of publication. For typing various drafts of the manuscript, we thank Sue
Leonard, Julie Meixelsperger, and James Poepsel. The drawings were done by Alice Prickett.
This work was supported by NIH grant HD-12659 to D.L.S. and NSF grant PCM 7903980
toJ.F.F.
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(Received 28 September 1981, revised 18 January 1982)