Download PDF

/. Embryol. exp. Morph. Vol. 67, pp. 13-25, 1982
Printed in Great Britain © Company of Biologists Limited 1982
13
The proportion and distribution
of polarizing zone cells causing morphogenetic
inhibition when coaggregated with anterior
half wing mesoderm in recombinant limbs
By JEANNE M. FREDERICK1 AND JOHN F. FALLON2
From the Department of Anatomy, The University of Wisconsin
SUMMARY
Previous work demonstrated that dissociated polarizing zone cells inhibit morphogenesis
when dispersed among dissociated anterior wing mesoderm cells in recombinant limbs. In
the present study the proportions and distribution of polarizing zone mesoderm which
inhibit morphogenetic expression in recombinant limb buds were determined. Completely
dissociated and pelleted anterior half wing mesoderm packed into leg ectodermal jackets
produced slender, digit-like outgrowths of bilateral symmetry in 60 % of the cases. This
represented the baseline with which recombinants containing polarizing zone cells were
compared. The inhibitory influence was first detected when polarizing zone cells constituted
16-18 % of the mesodermal component of recombinant limb buds; at this percentage the
incidence of distally complete outgrowths dropped to 23 % of the cases. The addition of
20-36 % polarizing mesoderm to dissociated anterior half wing gave reduced incidence (9 %)
of distally complete outgrowths. Percentages greater than 36 % polarizing zone cells led to
the total failure of distally complete limb-like morphogenesis in all cases. Further, when
more than 60 % polarizing mesoderm was dispersed with anterior half mesoderm, the outgrowths obtained were small, fleshy, and completely deficient in limb character. Finally, the
distribution of polarizing mesoderm in recombinant grafts was demonstrated to be random
at all percentages examined through 8 days, using chick/quail and autoradiographic methods.
In a separate set of experiments, flank mesoderm was not found to have the same inhibitory
effect on recombinant limb morphogenesis as described for polarizing mesoderm.
INTRODUCTION
The mesoderm along the posterior margin of the stage-17 to -28 (Hamburger
& Hamilton, 1951) chick wing bud has been demonstrated under experimental
conditions to influence polarity along the anteroposterior axis (Saunders &
Gasseling, 1968; A. B. MacCabe, Gasseling & Saunders, 1973; Tickle, Summerbell & Wolpert, 1975; Summerbell & Tickle, 1977; Fallon & Crosby, 1975a).
Tickle, et al. (1975) grafted polarizing mesoderm to sequential positions along
1
Author's present address: Cullen Eye Institute, Baylor College of Medicine, Houston,
TX 77030, U.S.A.
2
Author's address (for reprints): Department of Anatomy, The University of Wisconsin,
Madison, WI 53706, U.S.A.
14
J. M. FREDERICK AND J. F. FALLON
Dissociate
Dissect
I
Remove
ectoderm
Chick
donor
OO
Pellet
Anterior half
(nonpolarizing
mesoderm)
\
Recombinant,
graft to host
Chick
donor
Posterior
border
(polarizing
mesoderm)
Ectoderm al
jacket from
leg bud
Fig. 1. Scheme of tissue isolation and manipulation during the assembly of recombinant limb buds containing a known proportion of test cells (e.g. polarizing
mesoderm). For labelling studies, polarizing zone pieces were dissected from chick
donors previously injected with tritiated thymidine, or from quail donors.
the distal rims of stage-19 to -21 wing buds, from anterior to posterior borders,
and found that the digital pattern of duplicated structures induced was influenced by graft position along the anteroposterior axis. The polarity of the
duplications was determined by distance of the graft from the mapped region
(A. B. McCabe, et al. 1973) of polarizing activity. This suggested that cellular
cues related to anteroposterior polarity emanate from the polarizing region via
concentration of a diffusable substance, possibly a morphogen.
Asymmetries with respect to the anteroposterior axis disappear when
dissociated-reaggregated whole wing mesoderm is placed in limb ectodermal
jackets and allowed to grow (Zwilling, 1964). However, asymmetry can be
restored by including a small piece of polarizing mesoderm along a border of
such recombinants (J. A. MacCabe, Saunders & Pickett, 1973; Frederick, unpublished). Thus, addition of a small, intact block of polarizing mesoderm
produces improved morphogenesis by conferring anteroposterior polarity to
otherwise randomized limb mesoderm.
Anterior half wing mesoderm cells dissociated and assembled with ectodermal
jackets to form recombinant limbs yield good limb-like outgrowths. However,
the posterior half wing mesoderm cells in similar recombinants gives very poor
development, or none at all (J. A. MacCabe et al. 1973; Crosby & Fallon,
1975). If polarizing mesoderm was removed from posterior half wing buds and
Inhibition of recombinant limb morphogenesis
15
Table 1. Growth performance of recombinant? containing increasing
proportions of polarizing to nonpolarizing cells
% polarizing mesoderm added
to anterior half mesoderm
0%
10%
16-18%
20-22 %
25-29 %
30-34 %
36%
42%
47-52%
60-100 %
n=
n=
n=
n=
/*=
«=
n=
n=
n=
73
20
43
21
31
22
14
14
17
/i =
7
Limb-like
outgrowths
distally
complete
Limb-like
outgrowths
distally
incomplete
Small mound
or nothing
44(60-2%)
12(60-0%)
10 (23-2 %)
2 (9-5 %)
1(3-2%)
2(9-1%)
1 (7-1 %)
24(32-8%)
7(350%)
28(65-1%)
16(76-2%)
24(77-4%)
17 (77-2 %)
9(64-3%)
9 (64-3 %)
10(58-8%)
1(14-2%)
5 (6-8 %)
1(5-0%)
5(11-6%)
3 (14-2 %)
6(19-4%)
3(13-6%)
4(28-6%)
5 (35-7 %)
7(41-2%)
6 (85-7 %)
0
0
0
dissociated cell recombinants made from the remaining posterior mesoderm,
good limb-like development was achieved. Further, if dissociated polarizing
mesoderm is added to dissociated anterior mesoderm and recombinant limbs
made, these gave poor (i.e. lacking digit-like elements) or no development
(Crosby & Fallon, 1975). It was concluded that the dissociated and dispersed
polarizing mesoderm had an inhibitory effect on recombinant limb morphogenesis. This is in direct contrast with the inductive and polarizing capabilities
of the polarizing mesoderm in the other experimental conditions described
above. The aim of the present investigation was to determine the proportion and
distribution of polarizing zone cells that inhibit recombinant limb morphogenesis.
MATERIALS AND METHODS
Fertile White Leghorn chicken eggs and Japanese quail eggs were incubated
at 38 °C for 3^-4 days. Chick eggs were windowed according to the technique
of Zwilling (1959) and embryos of stages 21-22 used as donors. To rule out
mesodermal contamination of the ectodermal jacket, recombinant limb buds in
this study were assemblies of leg bud ectoderm with wing bud mesoderm.
Dissected anterior half wing mesoderm and polarizing zone pieces (Fig. 1)
were placed in 2 % 1:300 trypsin and 1 % pancreatin in Ca 2+ - and Mg2+-free
Hanks' balanced salt solution (CMF Hanks') for 18 min at 38 °C. These were
transferred to Hanks' balanced salt solution (Hanks' BSS) and the ectoderm
removed. The mesodermal pieces were rinsed for 5 min in CMF Hanks' at room
temperature, transferred to fresh CMF Hanks', and incubated for 15 min at
38 °C. They were then placed in 2 % 1:300 trypsin and 1 % pancreatin for
20 min at 4 °C, followed by 16 min in the same solution at 38 °C. This was
withdrawn and the fragments rinsed three or more times with a 1:1 mixture of
16
J. M. FREDERICK AND J. F. FALLON
Fig. 2. Recombinant grafts after 8 days of growth in ovo. (a) An example of a graft
which contained only dissociated anterior half wing mesoderm and gave limb-like
outgrowth, distally complete (Table 1). Note the long and slender proximal cartilage
model articulating with a short, phalangeal-like element; as is characteristic of
recombinants without polarizing mesoderm, these outgrowths are bilaterally
symmetrical about the anteroposterior axis, (b) Graft which contained 33 % polarizing zone cells included with dissociated anterior half wing mesoderm which formed
a limb-like outgrowth which was distally incomplete (Table 1). Ill-defined cartilages
(arrows), fused articulation points, distal deletions, and abundant soft tissue (arrow
heads) are typical of the distinctive, squat morphology occurring with substantial
proportions of polarizing mesoderm within a recombinant limb (c) Small cartilage
nodule (arrow) obtained from recombinant grafts containing 80% polarizing
mesoderm mixed with dissociated anterior half wing mesoderm and shown on host
embryo. Before clearing, this graft appeared as a small fleshy mound. Figs. 2 b and
2 c are the same magnification.
foetal calf serum and Hanks' BSS. A 1:2 mixture of foetal calf serum and
Hanks' BSS was added after the final rinse and the mesoderm dissociated by
triturating through a fine-bore pipette. Dissociation was completed by vortexing
at low speed for 5-10 seconds. The resulting mesodermal suspensions were
composed of >95 % single cells; the suspensions, of nonpolarizing (anterior
half) and polarizing cell populations were volumetrically combined after cell
counts for each were determined using a hemocytometer. Dead cells routinely
accounted for < 2 % of either cell population as judged by trypan blue exclusion.
The combined mesodermal suspension, vortexed to ensure adequate mixing of
cells, was incubated for 30 min at 38 °C. Cells were pelleted by mild centrifugation for 6 min followed by incubation for about 1 h at 38 °C. The pellet was
placed in 1:2 foetal calf serum and Hanks' for 15 min at room temperature in
preparation for recombinant assembly.
To prepare the ectodermal jackets, whole leg buds were put in CMF Hanks'
for 10 min, then transferred to 2 % trypsin and 1 % pancreatin in CMF Hanks'
for 3-4 h at 4 °C. After washing, the leg buds were added to the dish containing
the reaggregated mesodermal pellet. The ectoderm of each leg bud was removed
and saved. Recombinants were made and grafted to hosts as described in Crosby
& Fallon (1975). After 8 days, host embryos bearing the grafts were fixed in
10 % formalin and stained with Victoria blue for cartilage.
For histological examination, recombinants were harvested at 12 h intervals
Inhibition of recombinant limb morphogenesis
17
Fig. 3. Comparative light micrographs of recombinant limbs, (a) Section through
the apical ridge of recombinant limb containing 32 % polarizing zone cells, 60 h
after being grafted. Note that the ectoderm consists of simple cuboidal epithelium
overlain with a darkly stained periderm cell layer. The mesenchymal cells of the
mesoderm appear widely separated by extracellular ground substance, (b) Frontal
section of recombinant limb which contained 30 % polarizing zone cells, 72 h after
being grafted. Vascular degeneration is evident at this time, mesenchyme is riddled
with large sinus-like vessels, (c) Section through the apical ridge of recombinant
limb without polarizing zone cells, 66 h after being grafted. The ectoderm is apically
heightened in a pseudostratified columnar configuration. Debris-laden phagocytic
cells are particularly observed in the periderm layer, but this is not unusual. Note
the close packing of mesodermal cells and prominent nucleoli. (d) Frontal section
of recombinant limb without polarizing zone cells, 72 h after being grafted. Note
the fine calibre of the vascular pattern.
until 60 h following grafting. These recombinants were fixed in 0-02 % trinitrophenol, 2-0 % formaldehyde and 2-5 % gluteraldehyde in 0-075 M phosphate
buffer, postfixed in 1-0 % osmium tetroxide in 0-1 M 5-collidine, dehydrated and
embedded in Epon 812; sections 2/im thick were stained with methylene blue
and azure II (Fallon & Kelley, 1977).
Chick/quail xenoplastic recombinants and autoradiography of homoplastic
recombinants were used to assess the sorting pattern and the participation of
polarizing mesoderm in recombinant limb development. Quail polarizing
cells were distinguished in chick recombinants by the presence of one to
three strongly Feulgen-positive heterochromatin clumps within their nuclei
18
J. M. FREDERICK AND J. F. FALLON
Fig. 4. Recombinant grafts containing 17 % quail polarizing zone cells, (a) After
62 h w ovo, the randomly distributed quail cells are distinguished by darkly staining
nuclei. Micrograph from the centre of the recombinant. Frontal section, (b) After
62 h in ovo, the quail cells are seen on either side of the marginal vein at the middistal tip. Same section as (a), (c) After 8 days in ovo, small clusters of quail cells are
present in cartilage and some quail nuclei are distinguished in the perichondrium as
well. Frontal section, (d) After 8 days in ovo. In soft tissue areas, quail nuclei are
seen within aligned cells suggestive of myotubes (arrow); quail cells were often
observed in dermal papillae as well. Same section as (c).
Inhibition of recombinant limb morphogenesis
19
(LeDouarin & Barq, 1969). These xenoplastic recombinants were allowed to
grow from 1-3 days or up to 8 days, fixed, embedded in paraffin, sectioned,
and stained with the Feulgen reagent and fast green. Other xenoplastic recombinants allowed to grow for 8 days were stained with Victoria blue for cartilage.
In the homoplastic experiments, cells of polarizing mesoderm were 100 %
labelled with tritiated thymidine. These were included in recombinants containing unlabelled anterior half mesoderm. Recombinant limbs containing labelled
polarizing mesoderm were harvested and fixed at 16, 24, 48, and 72 h after
grafting and processed for autoradiography by standard methods (cf. Pollak &
Fallon, 1976).
RESULTS
Quantitation of the minimal proportion of polarizing mesodermal
cells producing morphogenetic inhibition
It was first necessary to establish a reference of morphogenetic performance
to which recombinants containing polarizing mesoderm were compared. When
recombinants of anterior half mesoderm were made, distally complete limb-like
outgrowths were obtained in 44 of 73 cases (60 %) (Fig. Id); this 60 % figure
represented the baseline of performance, i.e. results obtained under optimal
conditions when no polarizing mesoderm was present in recombinant limbs
(Table 1). The incidence of digits reported here may reflect recent modifications
of the protocol (cf. Crosby & Fallon, 1975; and Materials and Methods)
required to produce complete mesodermal dissociation of stage-21 to -22 wings.
The occurrence of distally complete limb-like outgrowths decreased as the
amount of dissociated polarizing mesoderm included with anterior half mesoderm increased (Table 1). When polarizing mesoderm comprised 16-18 % of
the mesodermal component of a recombinant bud, most of the resulting outgrowths were limb-like in appearance but were missing jointed phalangeal-like
structures in 28 of 43 (65 %) cases. Whereas dissociated anterior half alone
produced digit-like structures in 60 % of the cases, inclusion of 16-18 % polarizing mesoderm resulted in a reduction of digit-like outgrowths to 10 of 43 cases
(23%). Fewer limb-like outgrowths were able to be classified as 'distally
complete' due to deletion or fusion of distal phalangeal-like elements.
Increasing the concentration of polarizing zone cells to proportions between
20 and 36 % gave results that were similar, and for convenience, are discussed
together. In this percentage range, morphogenetic inhibition became more pronounced and two effects were observed: (1) deletion of distal elements, and (2)
reduction in size and discreteness of cartilagenous models that did form as
defined by the uptake of stain. A distinctive morphology became progressively
evident within this percentage range (Fig. 2 b). Cartilaginous models appeared
squat, compact, and were surrounded by more fleshy tissue than usual. This
type of outgrowth never appeared when dissociated anterior border mesoderm
was included with anterior half mesoderm in comparable proportions. For
20
J. M. FREDERICK AND J. F. FALLON
example, of 13 cases where anterior border (derived from quail) represented
15-42% of recombinant mesoderm, distally complete limb-like outgrowths
were obtained in 10 cases (77 %), with the remaining 3 (23 %) demonstrating
good, but imperfect skeletal patterns.
Where the proportion of dissociated polarizing mesoderm was above 36 %,
the outgrowths that occurred were terminally deficient or fused (Table 1).
Proportions above 60 % suppressed limb-like outgrowth almost completely
(Fig. 2 c). Of 38 cases in which polarizing mesoderm was included with anterior
half in excess of 36 %, no distally complete outgrowths resulted. Of 7 cases in
which polarizing mesoderm comprised 60-100 % of the recombinant mesoderm,
all were distally incomplete with 6 of the 7 yielding small cartilage nodules or
tufts of soft tissue only.
The specimens discussed above represent all grafts that, at 24-36 h postoperatively, were vascularized and whose hosts survived the 8-day growth
period. Although initially the graft may have appeared curled and white, by
12 h a typical graft became translucent and plump in appearance; proximal and
peripheral areas appeared translucent first while the centre remained opaque.
From combined histological and gross observation, it appeared that there was
a necrotic core which was cleared by ingestion of debris by phagocytes. At the
same time, a peripheral terminal blood vessel formed. A proximal arterial vessel
penetrated the graft centrally, and soon became continuous with the already
formed terminal vessel. There was a characteiistic avascular area beneath the
apical ridge. Subsequently, there seemed to occur a rapid proliferation of
mesodermal cells characterized in histological section at 48 h by numerous
mitotic figures, and elongation primarily along the proximodistal axis. Grafts
that contained even substantial proportions (e.g. 30 %) of polarizing mesoderm
were grossly indistinguishable at 48 h from those that did not contain polarizing
cells. However, between 2\ and 3 days, blood stasis, blood vessel enlargement,
and breakdown of the vascularization began in recombinants containing higher
proportions of polarizing mesoderm. By 3 days, very large sinus-like vessels
made up a significant part of such recombinants. The mesoderm was loosely
packed, and the apical ectodermal ridge had become simple cuboidal in
morphology (Figs. 3 a, b). In contrast, in recombinants having no polarizing
zone cells, blood vessels remained fine-calibered channels through which the
blood cells flowed. The mesodermal cells were tightly packed with prominent
nucleoli in their nuclei. The apical ectodermal ridge had the tall pseudostratified
columnar morphology seen in normal limb buds (Figs. 3 c, d).
The distribution of polarizing mesoderm within recombinant limbs
Autoradiography. When polarizing mesoderm comprised 20-36 % of a recombinant, distal deletions, fusion, and a squat morphology were characteristic
of resulting outgrowths. Therefore, 30 % was the proportion of tritiatedthymidine-labelled polarizing mesoderm chosen to observe the distribution of
Inhibition of recombinant limb morphogenesis
21
polarizing mesoderm in the recombinant. This percentage was beyond the
amount required to produce minimal inhibitory effects, and contained enough
cells to be easily visualized.
Of the recombinant grafts containing 30 % tritiated-thymidine-labelled
polarizing mesoderm, only the 24 h specimens warrant reporting in detail
because the label was diluted after this time. These cases (4) displayed a random
distribution of radioactive-thymidine-labelled cells (polarizing mesoderm) with
the original 30 % proportion being approximately maintained. Grafts harvested
at 48 and 72 h showed a random distribution of very lightly labelled cells.
Chick-quail. Results obtained using chick-quail xenoplastic recombinants
corroborated those of autoradiography with the advantage that the quail
Feulgen-positive marker does not suffer from successive dilution with cellular
proliferation. Therefore, the location of the marked cells could readily be discerned until they reached an advanced stage of differentiation. Quail posterior
border mesoderm possesses polarizing activity as assayed by grafting an intact
piece to the anterior border of a host chick wing (Fallon & Thorns, 1979).
Further, quail posterior border mesoderm is capable of inhibiting recombinant
outgrowth and morphogenesis when dissociated and mixed with cells of chick
anterior half mesoderm and follows the same pattern in Table 1. In a baseline
series (8 cases), the distribution of quail mesoderm cells derived from anterior
border was observed among cells of chick anterior half wing mesoderm in
recombinant limbs. Pockets of quail cells were randomly distributed within such
recombinants at all times sampled, using 15-17% and 42% quail anterior
border mixed with anterior half wing.
A total of 17 recombinant outgrowths containing up to 40 % quail polarizing
mesoderm was sectioned and examined. Of 5 recombinants containing 8 % quail
polarizing mesoderm, all demonstrated a random and sparse distribution of the
quail cells. At early times, i.e. up until 24 h, the quail cells were detected only
with careful examination since they most often appeared as isolated, individual,
intensely staining nuclei. At later times, however (e.g. 48-72 h and 8 days),
detection of quail cells was facilitated by the fact that they were present in
clusters, even though the clusters were few in number.
Recombinant limbs containing quail polarizing mesoderm in the 17-19 %
range (7 cases) were sectioned and examined at intervals until 8 postoperative
days. As depicted in Fig. 4, quail cells were evident in small, randomly scattered
pockets both at 62 h and 8 days. Quail cell clusters were never consistently
associated with any particular zone of the developing recombinant; rather, their
distribution was random. Quail cells, obtained from wing posterior border,
dissociated, and mixed with chick anterior half wing mesoderm, were found in
all mesodermal tissue types - dermis, hypodermis, muscle and its precursors,
and cartilage.
With inclusion of greater proportions of mesoderm derived from quail
posterior border, i.e. in the 34-40 % range (5 cases), at 58 and 82 h, the random
22
J. M. FREDERICK AND J. F. FALLON
disposition of quail cell pockets appeared to be maintained. However, at these
high percentages, there were enough quail cells present that boundaries between
the pockets became obscured.
The distribution and effect of flank cells on recombinant limbs
When dissociated flank mesoderm was tested in the same manner as cells of
the polarizing zone, comparable results were not obtained. Using 16 % dissociated flank cells, distally complete limb-like outgrowths occurred in 5 of 6 cases.
Anterior half wing mesoderm with 26-30 % dissociated flank mesodermal cells
yielded distally complete outgrowths in 8 of 19 (42 %) cases, distally incomplete
limb-like structures in another 8 cases, and small mounds or less in the remaining 3. Histological examination of grafts containing labelled (quail or radioactive chick) flank cells revealed that at 24 h the flank cells were randomly
distributed. However, it appeared that the original percentage was not maintained. Specifically, when 32 % tritiated-thymidine-labelled chick flank cells
were initially added to the mesodermal aggregate, only a small fraction of this
total amount could be visualized in recombinants harvested after 24 h of growth.
These few cells were invariably intensely labelled and located in the middle or
distal portion of the recombinant; this suggests that flank cells failed to proliferate in the recombinant limb and possibly were eliminated. Similarly, after
8 days of recombinant growth, quail flank cells were detected in proximal
dermal papillae and hypodermis only. Differences in the results using labelled
flank cells, which distinguish the persistent and random sorting behaviour of
polarizing region cells under similar conditions, are: (1) the absence of labelled
cells in 48 and 72 h grafts initially containing 32 % labelled flank cells, save for
a rare and intensely labelled cell; and (2) the fine calibre of distal vasculature of
72 h recombinant outgrowths.
DISCUSSION
The data reported in this paper demonstrate that polarizing zone cells will
cause detectable changes in the morphogenetic performance of recombinant
limbs at relatively low percentages. The deleterious effects increase as the
percentage of polarizing zone cells increase, leading ultimately to the complete
failure of recombinant outgrowth and morphogenesis. The use of cell markers
has demonstrated that the polarizing zone cells remain randomly distributed
throughout the 8-day development of the recombinant for all percentages tested.
The role of polarizing zone during normal limb development has not yet been
determined. Some investigators question the existence of such a zone or minimize its importance in any part of limb development (Saunders, 1977; Iten &
Javois, 1981). Others propose polarizing zone as the source of a diffusible
morphogen required at the time of the establishment of anteroposterior polarity
during the limb-bud stages of development (e.g. Summerbell, 1979; Tickle,
1980). Several investigators have advanced the hypothesis that polarizing zone
Inhibition of recombinant limb morphogenesis
23
may have a role in the initial induction of limb outgrowth and establishment of
its anteroposterior polarity (Fallon & Crosby, 19756, 1977; Smith, 1979;
Slack, 1979). This last possibility suggests the stabilization of mitotic rate in the
limb field (cf. Searls & Janners, 1971) which results in its outgrowth.
While there is no persuasive evidence for any of the various positions just
alluded to, it seems clear that the mesoderm of the posterior limb bud border
is a high point of morphogenetic activity. Under experimental conditions, this
zone has either stimulatory or inhibitory properties not found in any other part
of the limb bud. Qualitatively similar (stimulatory) activity has been found
elsewhere in the embryo (Saunders, 1977) viz. somite and flank. However, it is
always of reduced activity requiring a more sensitive assay than for the polarizing zone itself (Hornbruch, personal communication). We suggest it is more
than likely that the mechanism(s) involved with the action of the polarizing
zone are not unique to the developing limb. Rather, the posterior border may
be the first recognized polarizer in the embryo, and it is likely there are other
zones of such morphogenetic activity elsewhere. It is reasonable to assume that
the morphogenetic activity may diminish gradually from such high points. In
the case of the polarizing zone, the gradual decline would be what can be
measured in flank and somites by the more sensitive assay. It is worth stressing
that our study shows that flank cells do not have the inhibitory properties that
polarizing zone cells display.
There is another report of inhibition using dissociated-reaggregated mesodermal cells and recombinant limbs. Singer (1972) constructed recombinant
limbs composed of stage-30 leg chondrocytes combined with the stage-19
dissociated leg mesoblast. Graft size and perfection of distal structures improved
proportionally to the amount of stage-19 mesoblast included in the recombinant.
In a second series of experiments (Singer, 1972), stage-19 mesoblast cells were
combined with proximal limb-bud cells of stage 23, 24, or 25. Compared with
the stage-30 chondrocyte recombinants, these recombinants required more
stage-19 mesoderm to reach an equivalent stage of development. We point out
the fact that polarizing zone cells were included in the proximal limb mesoderm
used. Consequently, the inhibitory contribution of dissociated and dispersed
polarizing zone cells among nonpolarizing proximal limb and stage-19 mesoderm must be recognized in the analysis of the data and could account for the
increased amount of stage-19 mesoderm required for good development.
In a recombinant, mesodermal cells are randomized and simultaneously
polarity along the anteroposterior axis is abolished (J. A. MacCabe et al. 1973;
Zwilling, 1964). Polarity, or asymmetry can be restored by inclusion of an intact
piece of polarizing zone in the recombinant at the proximal and posterior
margin of the ectodermal jacket. Thus polarity can be reinstated in the potentially symmetric recombinant limb by polarizing zone. Recognizing that polarizing zone is located at one edge of the 3- to 6-day embryonic chick limb, it seems
reasonable that at some time during normal development this zone may specify
24
J. M. FREDERICK AND J. F. FALLON
'posterior' to those prospective limb cells capable of responding to its instruction. This instruction, or message, defines the posterior border, and is essentially
unidirectional. As already noted, the unidirectional signal also may be responsible for the initial outgrowth of the limb. We assume that dissociated,
single polarizing zone cells produce the same signal as polarizing zone collectively in a piece of posterior border mesoderm. An interpretation of the results
of these studies is that the persistence of random distribution of polarizing cells
within a recombinant produces a multidirectionality of message; cells capable
of responding in a prescribed manner to a given unidirectional signal fail to
respond properly when the message comes from many directions and there is
no clear concentration gradient along a single vector. This implies t h a t ' polarization' is a vector quantity having properties of direction and magnitude.
Indeed, there is evidence this is the case (Tickle, 1981). Alternatively, it is
possible that polarizing zone cells in a dissociated and dispersed condition may
produce an altered message. Although these are reasonable interpretations of
the data described in this report, further work must be done to determine how
polarizing zone cells can inhibit growth and differentiation of other limb cells
when dispersed in a recombinant, and stimulate growth and differentiation of
the same cells when grafted to the anterior border.
This investigation was supported by NSF Grant no. PCM7903980 and NIH Training
Grant no. T32HD7118. We thank Drs Allen W. Clark, David B. Slautterback, Ms Jeanie
Boutin, Ms Donene A. Rowe, and Ms B. Kay Simandl for their constructive criticism of the
manuscript. Special thanks are due Dr Gayle M. Crosby for her advice and assistance in the
early phases of this project. We thank Ms Lucy Taylor for the drawing and Ms Sue Leonard
and Mrs Julie Meixelsperger for typing the manuscript.
REFERENCES
G. M. & FALLON, J. F. (1975). Inhibitory effect on limb morphogenesis by cells of
the polarizing zone coaggregated with pre- or postaxial wing bud mesoderm. Devi Biol.
46, 28-39.
FALLON, J. F. & CROSBY, G. M. (1975a). The relationship of the zone of polarizing activity
to supernumerary limb formation (twinning) in the chick wing bud. Devi Biol. 42, 24-34.
FALLON, J. F. & CROSBY, G. M. (19756). Normal development of the chick wing following
removal of the polarizing zone. /. exp. Zool. 193, 449-455.
FALLON, J. F. & CROSBY, G. M. (1977). Polarizing zone activity in limb buds of amniotes.
In 'Vertebrate Limb and Somite Morphogenesis' (ed. D. A. Ede, J. R. Hinchliffe & M.
Balls), pp. 55-69. Cambridge: Cambridge University Press.
FALLON, J. F. & KELLEY, R. O. (1977). Ultrastructural analysis of the apical ectodermal ridge
during vertebrate limb morphogenesis. II. Gap junctions as distinctive ridge structures
common to birds and mammals. /. Embryol. exp. Morph. 41, 223-232.
FALLON, J. F. & THOMS, S. D. (1979). A test of the polar coordinate model in the chick wing
bud. Anat. Rec. 193, 534.
HAMBURGER, V. & HAMILTON, H. (1951). A series of normal stages in the development of the
chick embryo. /. Morph. 88, 49-92.
ITEN, L. & JAVOIS, L. (1981). Pattern regulation in the embryonic limb bud. Amer. Zool.
(In the press.)
LE DOUARIN, N. & BARQ, G. (1969). Sur l'utilisation des cellules de la Caille japonaise comme
'marquerurs biologiques' en embryologie experimentale. C.r. hebd. Seanc. Acad. Sci.,
Paris D 269, 1543-1546.
CROSBY,
Inhibition of recombinant limb morphogenesis
25
A. B., GASSELING, M. T. & SAUNDERS, J. W., JR. (1973). Spatiotemporal distribution of mechanisms that control outgrowth and anteroposterior polarization of the limb
bud in the chick embryo. Mech. Aging Develop. 2, 1-12.
MACCABE, J. A., SAUNDERS, J. W., JR. & PICKETT, M. (1973). The control of the anteroposterior and dorsoventral axes in embryonic chick limbs constructed of dissociated and
reaggregated limb-bud mesoderm. Devi Biol. 31, 323-335.
POLLAK, R. D. & FALLON, J. F. (1976). Autoradiographic analysis of macromolecular
syntheses in prospectively necrotic cells of the chick limb bud. II. Nucleic acids. Expl Cell
Res. 100, 14-22.
SAUNDERS, J. W., JR. (1977). The experimental analysis of chick limb bud development. In
Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls),
pp. 1-24. Cambridge: Cambridge University Press.
SAUNDERS, J. W. & GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in the
origin of limb symmetry. In Epithelial-Mesenchymal Interactions (ed. R. Fleischmajer &
R. E. Billingham), pp. 78-97. Baltimore: Williams and Wilkins.
SEARLS, R. L. & Janners, M. Y. (1971). The initiation of limb bud outgrowth in the embryonic
chick. Devi Biol. 24, 198-213.
SINGER, R. H. (1972). Analysis of limb morphogenesis in a model system. Devi Biol. 28,
113-123.
SLACK, J. (1979). Pattern formation in the chick limb bud. Nature 279, 583-584.
SMITH, J. C. (1979). Evidence for a positional memory in the development of the chick wing
bud. J. Embryol. exp. Morph. 52, 105-113.
SUMMERBELL, D. (1979). The zone of polarizing activity: evidence for a role in normal chick
limb morphogenesis. J. Embryol. exp. Morph. 50, 217-233.
SUMMERBELL, D. & TICKLE, C. (1977). Pattern formation along the antero-posterior axis of
the chick limb bud. In Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R.
Hinchliffe & M. Balls), pp. 41-53. Cambridge: Cambridge University Press.
TICKLE, C. A., SUMMERBELL, D. & WOLPERT, L. (1975). Positional signalling and specification
of digits in chick limb morphogenesis. Nature 254, 199-202.
TICKLE, C. (1980). The polarizing region and limb development. In Development of Mammals
(ed. M. H. Johnson), vol. 4, pp. 101-136. Amsterdam: Elsevier/North Holland Biomedical
Press.
TICKLE, C. (1981). The number of polarizing region cells required to specify additional digits
in the developing chick wing. Nature 289, 295-298.
ZWILLING, E. (1959). A modified chorioallantoic grafting procedure. Transplant Bull. 6,
238-247.
ZWILLING, E. (1964). Development of fragmented and dissociated limb bud mesoderm. Devi
Biol. 9, 20-37.
MACCABE,
{Received 22 April 1981, revised 20 July 1981)