/. Embryol exp. Morph. Vol. 53, pp. 67-73, 1979
Printed in Great Britain © Company of Biologists Limited 1979
(fj
The target tissue of limb-bud polarizing activity in
the induction of supernumerary structures
By JEFFREY A. MACCABE 1 AND BRENDA W. PARKER 1
From the Department of Zoology, The University of Tennessee
SUMMARY
When polarizing mesoderm from the posterior border of the 4-day chick limb bud is placed
adjacent to anterior limb mesoderm and ectodermal ridge, the anterior ridge thickens and
mesodermal outgrowth ensues, resulting in supernumerary limb structures. This apposition
of anterior and posterior limb tissues can be accomplished by cutting off the apical one third
of the limb bud and reimplanting it on the stump with its anteroposterior axis reversed. The
preaxial response to polarizing activity can be obtained after only 12-18 h in the reoriented
position. Reversed apical mesoderm develops supernumerary digits when combined with
untreated ectoderm. The reciprocal combination, reversed ectoderm and untreated mesoderm,
fails to develop supernumerary structures. We have interpreted this as evidence that, in
inducing supernumerary limb structures, polarizing activity acts only on the mesoderm.
INTRODUCTION
A region of polarizing activity has been described in embryonic limb-bud
mesoderm which has its highest level of activity along the posterior border. The
activity is characterized by its ability to induce supernumerary structures from
the preaxial region of the limb bud. This can be done in a number of ways
(Saunders & Gasseling, 1968), for example, by transplanting small blocks of
tissue from the posterior to the anterior border or by excising the apical one
third of the limb bud and reimplanting it on the stump with its anteroposterior
(a-p) axis reversed. The supernumerary structures that result are polarized along
their a-p axis and their posterior borders develop facing the source of the
polarizing activity.
Both the preaxial mesoderm and ectoderm respond to polarizing activity, the
ectodermal ridge by increasing in thickness and the mesoderm by outgrowth.
Whether the ectoderm and mesoderm are both responding directly to polarizing
activity, or whether this activity acts on one of these tissues which in turn acts
on the other, sequentially, is the question the experiments reported here address.
1
Authors' address: Department of Zoology, The University of Tennessee, Knoxville,
Tennessee 37916, U.S.A.
68
J. A. MACCABE AND B. W. PARKER
MATERIALS AND METHODS
The fertile White Leghorn chicken eggs used in these experiments were
obtained from the Department of Animal Science at the University of Tennessee,
Knoxville. The eggs were incubated in a forced-draft incubator at 37-5 °C and
55-60 % relative humidity. On the third day of incubation 2 c.c. of albumin were
removed from each egg and a window cut into the side of the shell. The windows
were then covered with Parafilm (American Can Co,) and the embryos returned
to the incubator until they reached stage 21-22 (Hamburger & Hamilton, 1951)
on the following day.
Recombinant limb buds were constructed by combining the ectoderm from
one limb with the mesoderm from another. The ectoderms were isolated after
a 2-^—3 h incubation in 1 % trypsin at 0-5 °C and mesoderms after a 25 min
incubation in 1 % EDTA (Ethylenedinitrilo-tetracetate, disodium) at 37 °C.
Both solutions were made up in calcium-magnesium-free Tyrode's saline. The
isolated tissues were combined at 0-5 °C in a 2:1 solution of Tyrode's and horse
serum. The recombinant limbs were allowed to warm to room temperature and
were then transplanted to the somites of host embryos.
In one series of experiments ectoderms were combined with mesoderms from
the opposite side of the embryo so that their a-p polarities were opposed but
their dorsoventral and proximodistal axes were matching. These recombinant
wing buds, with reversed ectodermal a-p polarity, were excised at intervals after
the host embryos were returned to the incubator, examined and either photographed or drawn with a camera lucida in order to record changes in the
appearance of the ectodermal ridge. After several attempts to measure ridge
thickness in these experimental limbs, we decided to subjectively evaluate
thickness in terms of whether the anterior or posterior half appeared thicker or
whether the ridge appeared uniformly thick. Measurements of ridge thickness
often gave misleading results because of irregularities in thickness that appeared
after tissue recombination, because of the continuously variable nature of ridge
thickness under most conditions and because of slight variations in the position
of the ectoderm on the mesoderm after recombination. A subjective evaluation
thus seemed to give us a reasonable approximation of ridge behavior.
In a second series of experiments the apical thirds of wing buds were excised
and pinned back onto their stumps with the polarity of the antero-posterior
axis reversed. After 45 min to 1 h at room temperature, the glass pins were
removed and the embryo returned to the incubator for 18 h. The reoriented
apices were then cut off slightly distal to the plane of the original excision and
used as ectoderm or mesoderm donors for tissue recombination. A slight notch
in the anteriorand posterior limb borders where the apex healed back onto the
stump was used as an indicator of the original plane of excision.
For autoradiography, embryos incubated for 3 days were given 10 //Ci of
[3H]thymidine (New England Nuclear, sp. act. 5 Ci/mmole) in 0-1 ml of Tyrode's
Target tissue of limb-bud polarizing activity
69
0-1 mm
Fig. 1. (A) Dorsal view of a wing bud J8 h after combining a stage-21 left mesoderm
with a right ectoderm and transplanting to a host. The a-p polarity of the ectoderm
was reversed on the mesoderm. Note the preaxial extent of the apical ridge (arrow)
compared to the normal stage-21 left wing (B).
saline by dropping the solution directly onto the embryo through the window in
the shell. The following day the apical one third of the wing bud was excised and
transplanted in reversed a-p orientation to an unlabeled host stump whose limb
apex had been excised and discarded. After 18 h of incubation the labeled apices
were cut off slightly distal to the original plant of excision and fixed in Carnoy's
solution for autoradiography. The stumps were also excised from the body wall
and fixed for autoradiography. Both the stumps and apices were embedded in
parafin and sectioned 8 jtim thick. The parafin was removed with toluene, the
sections hydrated, rinsed twice in 5% TCA and air dried. The slides were
dipped in Kodak NTB-2 liquid emulsion, air dried and stored for 3 weeks at
0-5 °C. The emulsion was developed in Kodak D-19 developer and the sections
stained in Mallory-Heidenhain stain.
RESULTS
One hundred and twenty-six tissue recombinations were made between isolated,
but otherwise normal, limb ectoderm and mesoderm with the ectodermal a-p
polarity reversed on the mesoderm. The polarity of ectodermal ridge thickness,
normally thinnest preaxially and thickest postaxially, was thus reversed after
recombination. After 12 h this was still true in nearly all cases. After 15 h,
however, the postaxial ridge of almost half (9/20) the recombinant limbs
appeared as thick or thicker than preaxial ridge. By 18 h over half (13/22) the
70
J. A. MACCABE AND B. W. PARKER
Table 1. Limb-bud mesoderm-ectoderm recombination
after apical reorientation
Total
Number with
number of supernumerary
recombinants
digits
Reoriented mesoderm + normal
ectoderm
Normal mesoderm + reoriented
ectoderm
31
19
29
0
postaxial ridges appeared thickened (Fig. 1). By 24 h after recombination nearly
all of the limbs appeared to have uniformly thickened ectodermal ridges. This
was also true after 36 and 48 h, but the entire ridge had become somewhat
thinner, as it normally does at this stage, making a gradient of ridge thickness
more difficult to visualize.
Five additional hosts for recombinant wing buds were not sacrificed until
7 days after transplantation. As expected (Zwilling, 1956), the fully developed
recombinant wings were normal and had antero-posterior polarity corresponding
to the mesodermal component of the recombinant bud.
Sixty tissue recombinations were performed between normal donor apices and
those that had been reoriented on their stump for a period of 18 h (Table 1).
In 29 cases normal apical mesoderms of stages 21-22 were combined with
ectoderm from reoriented apices. All of these developed into normal wing tips
after transplantation to hosts (Fig. 2A). In 31 instances, normal ectoderm of
stages 21-22 was combined with mesoderm from reoriented apices. Nineteen
of these developed with supernumerary preaxial (relative to the original
orientation of the apex) digits (Fig. 2B). Ten of these were supernumerary digit
Ills with reversed a-p polarity indicated by the distribution of feather follicles.
The remaining nine supernumerary digits corresponded to digit II of the wing.
The apices used in the experiments described above were excised after 18 h
in the reoriented position. This excision was made slightly distal to the original
excision, indicated by a small notch on the anterior and posterior borders where
the stump and apex healed together. As a control for contamination of the apex
by stump cells, an autoradiographic analysis was done in 12 experiments where
an [3H]thymidine-labeled apex was reoriented on an unlabeled stump and
excised in the same manner after 18 h. These experiments verified that there
was no stump tissue in the apex after the second excision. In all cases a thin layer
of labeled cells from the apex remained on the stump after the second excision.
Thus the preaxial side of the apex was exposed to polarizing activity for 18 h,
but contained no postaxial tissue.
Target tissue of limb-bud polarizing activity
71
1 0 mm
Fig. 2. (A) A normal wing tip that developed after combining mesoderm from an
unoperated apex with an ectoderm from an apex that had been reversed for 18 h.
(B) A supernumerary apex (arrow) that developed after combininga normal ectoderm
with mesoderm from an apex that had been reversed for 18 h.
DISCUSSION
Models accounting for the establishment of morphogenetic fields on the basis
of gradients were popular in the first half of this century. Subsequently, interest
in them waned, largely for lack of evidence for their existence. In the past
decade interest in gradient models has been renewed (Wolpert, 1971) and a
diffusion gradient model has been suggested to account for the establishment of
a-p polarity in the developing vertebrate limb (Wolpert, 1969). Saunders &
Gasseling (1968) demonstrated a mesodermal region in the posterior limb bud
which possesses 'polarizing activity'. It is this region which is thought to be the
source of the morphogen which establishes a-p polarity. Since these contributions by Saunders and Wolpert, considerable evidence has accumulated for a
diffusion gradient model. There is, for example, evidence for the existence of a
gradient along the a-p axis of the limb (MacCabe & Parker, 1975,1976) and for
a relationship between the distance from the source of the morphogen and the
a-p level specified (Tickle, Summerbell & Wolpert, 1975). There are also indications that this gradient is required for the development of a polarized a-p axis
(MacCabe, Lyle & Lence, 1979). While there is no direct evidence that the
morphogen is moving through the limb by diffusion, there may be a readily
diffusible substance involved (Calandra & MacCabe, 1978).
The nature of the response to this morphogen has not been well investigated.
In vivo the response includes mesodermal outgrowth as well as apical ectodermal
ridge thickening. The interdependence between ectodermal ridge and subjacent
mesoderm has long been known (Zwilling, 1961) and it is this relationship that
polarizing activity largely affects (Summerbell, 1974). Does the polarizing
mesoderm act on distal mesoderm, ectodermal ridge or both? Amprimo has for
some years emphasized the importance of interaction between proximal and distal
72
J. A. MACCABE AND B. W. PARKER
mesoderm (Amprino & Camosso, 1959; Amprino, 1968). Based upon the
observation that polarizing mesoderm cannot maintain a thickened ectodermal
ridge when placed immediately beneath it, Saunders & Gasseling (1968)
suggested that polarizing activity acts on the distal mesoderm.
The experiments reported here provide additional evidence that polarizing
activity acts directly on the mesoderm in the induction of supernumerary limb
structures. When the a-p polarity of the ectoderm (and thus the ectodermal
ridge) is reversed on the mesoderm, the thin ectodermal ridge overlying the
postaxial half of the mesoderm thickens in about 18 h. However, when the
polarity of both mesoderm and ectoderm are reversed, by reorienting the entire
limb apex, ridge thickening is not seen after 13-18 h, but is after 32-40 h
(Camosso & Roncali, 1968). The increased time it takes for the ridge to thicken
when the apical mesoderm, as well as ectoderm, is reoriented may represent the
time required for movement of the morphogen into the mesoderm and possibly
for some kind of mesodermal response. It seems logical to assume that this time
may be approximated by a determination of the minimum length of time the
apex must be in. the reoriented position to obtain supernumerary structures.
Saunders & Gasseling (1963) found this to be about 12 h, an interval consistent
with the timing discussed above.
More direct evidence that the polarizing factor acts on limb mesoderm was
obtained in our experiments involving apical reorientation followed by tissue
recombination. The apex was reoriented for 18 h (thereby exposing preaxial
tissue to polarizing activity) then excised and either the ectoderm or mesoderm
combined with normal apical mesoderm or ectoderm to make a recombinant
limb bud. Supernumerary structures developed only when reoriented mesoderm
was combined with normal ectoderm. The reciprocal combination, normal
apical mesoderm with reoriented ectoderm, gave rise to wing tips without supernumerary structures. The experiments provide clear evidence that the polarizing
morphogen affects the mesoderm directly, and the ectoderm only indirectly via
the mesodermal response. This response, the nature of which is unknown,
presumably gives rise to the altered interaction between ectodermal ridge and
mesoderm that results in the development of supernumerary limb structures.
This research was supported by NIH Grant HD 07282 and NIH Research Career Development Award HD 00228 to J.A.M.
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{Received 3 April 1979, revised 5 June 1979)