/ . Embryol. exp. Morph. Vol. 35, 2, pp. 369-381, 1976
Printed in Great Britain
369
Effect of dorsal and ventral limb
ectoderm on the development of the limb
of the embryonic chick
By ROBERT L. SEARLS 1
From the Department of Biology,
Temple University, Philadelphia
SUMMARY
Limb ectoderm plus a small amount of subjacent mesoderm obtained from, the dorsal
or the ventral surface of each of the four limbs of the chick embryo were grafted to the
dorsal surface of the right wing in either reversed or normal orientation. The host wings
developed an abnormal humerus in all cases except when the graft was obtained from the
dorsal surface of the right wing and was in normal orientation. However, the nature of the
abnormalities varied with the source of the graft and with the orientation of the graft. It
is unlikely that the abnormalities were related solely to the small amount of mesoderm
grafted since previous experiments have demonstrated that the mesoderm would regulate.
Flank ectoderm plus a small amount of flank mesoderm did not cause limb abnormalities
when grafted in the same manner.
INTRODUCTION
It has been proposed that the dorsal and ventral ectoderm of the embryonic
chick wing play a significant role in normal wing development (Pautou &
Kieny, 1973; Stark & Searls, 1974; MacCabe, Errick & Saunders, 1974). A
block of mesoderm from the elbow region of the wing of a stage 21-23 embryo
(Hamburger & Hamilton, 1951) that had been rotated 180° and inserted back
in the elbow region tended to regulate if the ectoderm had been removed.
If the dorsal and ventral ectoderm were rotated with the mesoderm, at stage 23
the mesoderm did not regulate and at stages 21 and 22 the humerus was
shorter than normal and had a spur of ectopic cartilage perpendicular to the
long axis of the humerus. When only dorsal ectoderm with a small amount
of subjacent mesoderm was rotated 180°, the humerus was short and had
a prominent spur (Stark & Searls, 1974). The dorsal and ventral ectoderm
may control also the dorsoventral axis of the limb (Pautou & Kieny, 1973;
MacCabe et al. 1974). When a limb ectodermal jacket was placed on a limb
mesoblast with an orientation other than that of the mesoblast, proximal
structures developed with the orientation of the mesoblast but more distal
structures developed with the dorsoventral orientation of the ectoderm.
1
Author's address: Department of Biology, Temple University, Philadelphia, Pa. 19122,
U.S.A
24
EMB 35
370
R. L. SEARLS
The present experiments were initiated to investigate further the influence
of polarity of the ectoderm on normal limb development. Limb ectoderm with
a small amount of subjacent mesoderm that had been obtained from the
dorsal and ventral surfaces of each of the four limbs was grafted to the dorsal
surface of the right wing in both normal and reversed proximal-distal
orientation. Dorsal right-wing ectoderm grafted to the dorsal right wing in
normal orientation has all axes normal while in reversed orientation has both
the proximal-distal and cranial-caudal (anterior-posterior) axes reversed.
Ventral right-wing ectoderm grafted to the dorsal surface of the right wing
in normal proximal-distal orientation has its cranial-caudal axis reversed
while in reversed proximal-distal orientation has its cranial-caudal axis normal
(and is ventral ectoderm rather than dorsal). Only dorsal right-leg ectoderm
is a dorsal ectoderm with the same axes as dorsal right wing ectoderm, and
still is different because it is leg rather than wing.
It has been found that only ectoderm with a small amount of subjacent
mesoderm from the dorsal surface of the right wing grafted in normal orientation
routinely produces normal wings. Ectoderm from all other sources grafted in
either normal orientation or reversed orientation produced abnormal development of the proximal region of the wing. However, the way in which the
proximal region of the wing was abnormal varied with the source of the
ectoderm and the orientation of the ectoderm.
MATERIALS AND METHODS
Fertilized White Leghorn eggs (Shaw's Hatchery; West Chester, Pa.) were
incubated at 37 °C. Windows were cut in the eggs during the third day of
incubation (Zwilling, 1959), and the eggs were returned to the incubator to
develop to the desired stage.
A donor embryo (stage 22-23) was placed in a dish of Tyrode's solution
and its limbs excised. The limbs were placed in separate dishes of Tyrode's
solution, and were used in turn as source of tissue to be grafted. The base
of the limb was stained with 2 % nile blue sulfate in 2 % agar (Hamburger,
1960). The dorsal and ventral proximal regions were isolated surgically (Searls,
1967) as roughly triangular pieces 0-4-0-6 mm on a side and 0-1-0-15 mm
thick. The distal-caudal corner of each region was stained with nile blue
sulfate. The staining of the proximal edge and of the distal caudal corner, plus
the shape of the piece, permitted the ectoderm plus subjacent mesoderm to be
oriented. These pieces were grafted into the right wing of host embryos as
soon as they were prepared.
Host embryos were stage 22-24. Working through a window in the shell, a
sheet of dorsal ectoderm plus a minimum of subjacent mesoderm was loosened
and removed from the dorsal surface of the right wing using a fine tungsten
needle. Ectoderm plus subjacent mesoderm from the donor embryo was
Effect of limb ectoderm
371
grafted into this site. The window was resealed and the egg was incubated
at 37 °C. Many of the grafts were inspected 18-24 h after the operation to
determine if the graft had taken to the host wing. However, loss of the graft
could not always be detected in this manner; wings that appeared to carry
a graft by inspection through the window in the shell were found to have
lost the graft when they were removed to a dish of Tyrode's solution. Since
the loss of the graft could not always be detected by inspection, and inspection
seemed to increase the number of host embryos that died, inspection of the
host wings 24 h after the operation was discontinued during the later stages
of these experiments. After 7 days of further development, both the right and
left wings were fixed in 10 % formalin, stained in methylene blue according
to the method of Lundvall (Hamburger, 1960), and cleared in methyl salicylate.
RESULTS
The results of these grafting experiments are summarized in Tables 1 and 2.
The results obtained from each kind of graft were highly variable, but in
every case only the proximal regions were changed; the regions distal to the
elbow were always normal. The humerus was often short and the proximal
region often contained ectopic cartilage. The ectopic cartilage was sometimes
present as a rod of cartilage perpendicular to the humerus (this will be called
a 'spur'), sometimes as a rod of cartilage parallel with the axis of the humerus
(this will be called 'duplicate'), sometimes the humerus was of approximately
normal thickness on one axis but wider than normal on another axis (this
will be called 'wide'), and sometimes the ectopic cartilage was present as
cartilaginous nodules (this will simply be called ectopic cartilage). Every
Table 1. Ectoderm plus mesoderm grafted in normal
proximal-distal orientation
Source
No.
No.
normal
No. with
short
humerus
Spur*
Widef
Ectopic
cartilage^
Dorsal right wing
11
0
0
0
3
8
0
0
2
Ventral right wing
12
3
8
0
2
4
Dorsal left wing
13
5
5
0
0
0
Ventral left wing
9
4
5
3
3
3
Dorsal right leg
8
2
0
2
10
1
1
Ventral right leg
7
0
0
8
0
Dorsal left leg
11
0
11
0
2
5
8
Ventral left leg
14
0
7
33
14
Total
88
23
28
* Ectopic cartilage essentially perpendicular to the humerus, cf. Figs. 2-4.
f Humerus wide and thin, cf. Fig. 5.
% Ectopic cartilage in the vicinity of the humerus that cannot be combined into any single
category.
24-2
ill
R. L. SEARLS
Table 2. Ectoderm, plus mesoderm in reversed proximal-distal orientation
No. with
short
normal humerus
No.
Source
No.
Spur*
Widef
Duplicatej
Ectopic
cartiIage§
1
3
13
2
6
7
0
Dorsal right wing
4
6
0
9
2
0
5
Ventral right wing
1
9
0
9
0
4
6
Dorsal left wing
4
7
0
4
1
2
Ventral left wing
2
1
3
4
7
2
1
Dorsal right leg
2
7
11
2
6
0
1
Ventral right leg
1
11
7
8
0
0
3
Dorsal left leg
1
2
4
3
3
9
0
Ventral left leg
Total
21
76
46
14
29
5
11
* Ectopic cartilage essentially perpendicular to the humerus, cf. Figs. 6, 7, 9.
t Humerus wide and thin, cf. Fig. 10.
% Ectopic cartilage essentially parallel with the humerus, cf. Fig. 8, 10 and 11.
§ Ectopic cartilage in the vicinity of the humerus that cannot be combined into any
single category.
combination of these was obtained: a few limbs had a short humerus with a rod of
ectopic cartilage perpendicular to the humerus, another rod parallel with the
humerus, the humerus was wide on one axis, and further nodules of cartilage
were present near the humerus. It proved impossible to make a table in which
every combination could be listed separately. The number of limbs that were
abnormal in several ways will be mentioned in the description of each kind
of graft when it seems to be important.
Grafts in normal proximal-distal orientation
Wings carrying grafts from the dorsal surface of the right wing-bud in
normal proximal-distal orientation did not produce ectopic cartilage (Table 1). In
three out of 11 such wings (27 %) the humerus was normal in shape but shortened
about 25 % relative both to the length of the humerus in the contralateral
control wing and to the length of the radius and ulna in the experimental
wing (Fig. 1).
Wings carrying grafts in normal proximal-distal orientation from the ventral
surfaces of the right wing, left wing, right leg, left leg, and from the dorsal
surface of the left wing were often abnormal (40 out of 58; 69 %) (Table 1).
Many of these had a spur of cartilage perpendicular to the humerus (33 out of
58; 57 %). The spur was always at the base of the wing and attached to either
the caudal (Figs. 2, 3) or to the ventral (Fig. 4) surface of the humerus. The
size and shape of this spur of cartilage varied but not in a manner that could
be easily related to the source of the graft. The humerus was short in only eight
out of 58 (14 %) of these wings; five of the wings with a short humerus also
had a spur while three had no abnormality other than a short humerus.
Effect of limb ectoderm
373
Wings carrying grafts in normal proximal-distal orientation from the dorsal
surface of the right and left legs usually had an abnormal humerus (17 out of
19; 90 %). However, instead of a spur of ectopic cartilage these wings frequently
had a wide humerus (13 out of 19; 68 %) (Table 1). Inspection of these wings
suggested that the humerus had formed as a plate on the same plane as the
radius and ulna. In some cases the plate appeared to be almost perpendicular
to the long axis of the embryo and to the plane of the radius and ulna, but
this appeared to be due to a rotation of the humerus sometime between the
formation of the humerus and 11 days of embryonic development. In a few
cases the plane of the radius and ulna appeared to have rotated with the
rotation of the humerus (Fig. 5). The humerus was short in 11 out of 19 (58 %)
of these wings; 10 of the wings had a humerus that was both wide and short.
Only one wing had a short but otherwise normal humerus.
Grafts in reversed proximal-distal orientation
Wings carrying grafts from the dorsal surface of the right wing in reversed
proximal-distal orientation were usually abnormal (11 out of 13; 85%)
(Table 2). All of the abnormal wings had ectopic cartilage, usually (seven out
of 13; 54 %) as a spur at the base of the humerus (Fig. 6) or more distally along
the humerus (Fig. 7). Many of the wings also had a short humerus (six out of
13; 46%). These results are similar to those reported earlier for experiments
in which ectoderm plus subjacent mesoderm on the dorsal surface of the wing
was loosened, rotated 180°, and grafted back into the dorsal surface of the
wing (Stark & Searls, 1974) except that in the previous experiments 70 % were
short and 38 % had ectopic cartilage. In a few cases (three out of 13; 23 %),
wings carrying grafts from the dorsal surface of the right wing in reversed
proximal-distal orientation had ectopic cartilage parallel with the humerus;
this had not been observed in the previous experiments.
Wings carrying grafts from the ventral surfaces of the right and left wings
and from the dorsal surface of the left leg in reversed proximal-distal orientation
were often abnormal (20 out of 27; 74%) (Table 2). These wings frequently
had a wide humerus (17 out of 27; 63 %), and in addition many had ectopic
cartilage parallel with the humerus (12 out of 27; 44 %) (Fig. 8). None of these
wings had a spur of cartilage perpendicular to the humerus. Of the 20 wings
with ectopic cartilage, 18 had a short humerus.
Wings carrying grafts from the dorsal surface of the left wing and from
the dorsal and ventral surfaces of the right leg in reversed proximal-distal
orientation had a range of abnormalities; only two out of 27 (7 %) were normal
(Table 2). All other wings (25 out of 27) had ectopic cartilage. Sometimes the
humerus was wide with ectopic cartilage present either as a spur or parallel
with the humerus. Occasionally both a spur and parallel cartilage was observed
together with a short, wide humerus (Figs. 9, 10). In many cases (21 out of
25 wings) a short humerus was observed in addition to ectopic cartilage.
374
R. L. SEARLS
Effect of limb ectoderm
375
Wings carrying grafts from the ventral surface of the left leg in reversed
proximal-distal orientation were all abnormal but six out of nine were of
normal length. Two of these wings contained a humerus of normal length but
wide, and three had a humerus of normal length but with cartilage parallel
with the humerus (Fig. 11). The other four wings all had ectopic cartilage of
various kinds and three of the other four were short.
FIGURES
1-8
Fig. 1. Ectoderm plus some subjacent mesoderm from the dorsal surface of the
right wing of a stage-24 embryo grafted into the dorsal surface of the right wing
of a stage-23 embryo in normal proximal-distal orientation. The length of the
humerus of the right wing is 77 % of the length of the humerus of the left wing
and 71 % of the length of the ulna of the right wing. The humerus is normal other
than in length, x 6.
Fig. 2. Ectoderm plus some subjacent mesoderm from the ventral surface of the
right leg of a stage-22+ embryo grafted into the dorsal surface of the right wing
of a stage-23 embryo in normal proximal-distal orientation. The length of the
humerus of the right wing is 92 % of the length of the humerus of the left wing
and 94 % of the length of the ulna of the right wing. The arrow points to a spur
of ectopic cartilage extending from the caudal surface of the humerus. x 6.
Fig. 3. Same as Fig. 2 at higher magnification to show more clearly the spur of
ectopic cartilage, x 11.
Fig. 4. Ectoderm plus some subjacent mesoderm from the ventral surface of the
right wing of a stage-22+ embryo grafted into the dorsal surface of the right
wing of a stage-22+ embryo in normal proximal-distal orientation. The length
of the humerus is 92 % of the length of the ulna. The arrow points to a spur of
ectopic cartilage extending from the ventral surface of the humerus. x 9.
Fig. 5. Ectoderm plus some subjacent mesoderm from the dorsal surface of the
right leg of a stage-24 embryo grafted into the dorsal surface of the right wing of
a stage-23 embryo in normal proximal-distal orientation. The length of the
humerus is 53 % of the length of the ulna. Photographed from the ventral and cranial
aspect (note the tips of forceps holding down the scapula). The articulations of
the radius and ulna are in the same plane as the humerus; the radius and ulna
are not in their normal plane, x 9.
Fig. 6. Ectoderm plus some mesoderm from the dorsal surface of the right wing
of a stage-23 embryo grafted into the dorsal surface of the right wing-bud of a
stage-23 embryo in reversed proximal-distal orientation. The length of the humerus
is 50 % of the length of the ulna. The arrow points to a spur of ectopic cartilage
proximal on the humerus. x 12.
Fig. 7. Ectoderm plus some mesoderm from the dorsal surface of the right wing
of a stage-22 + embryo grafted into the dorsal surface of the right wing of a stage-22 +
embryo in reversed proximal-distal orientation. The length of the humerus is
58 % of the length of the ulna. The arrow points to a spur of ectopic cartilage distal
on the humerus. x 12.
Fig. 8. Ectoderm plus some mesoderm from the ventral surface of the right wing
of a stage-22 + embryo grafted into the dorsal surface of the right wing of a
stage-22— embryo in reversed proximal-distal orientation. The length of the
humerus is approximately equal to the length of the ulna. The arrow points to
ectopic cartilage parallel with the humerus. x 11.
376
R. L. SEARLS
Fig. 9. Ectoderm plus some mesoderm from the dorsal surface of the right leg of
a stage-22+ embryo grafted in the dorsal surface of the right wing of a stage-22 +
embryo in reversed proximal-distal orientation. The length of the humerus is
75 % of the length of the ulna. The arrow points to a spur of ectopic cartilage
perpendicular to the humerus. Photographed from the dorsal aspect; notice that
the radius and ulna lie on a plane perpendicular to their normal plane, x 13.
Fig. 10. The same wing as in Fig. 9 photographed from the caudal aspect. The
arrows point to ectopic cartilage approximately parallel with the humerus and to
ectopic cartilage in the vicinity of the elbow. Notice that the radius and ulna lie
on a plane perpendicular to their normal plane, x 13.
Fig. 11. Ectoderm plus some mesoderm from the ventral surface of the left leg of
a stage-22+ embryo grafted in the dorsal surface of the right wing-bud of a
stage-23 embryo in reversed proximal-distal orientation. The length of the humerus
is 100% of the length of the ulna. The arrow points to ectopic cartilage parallel
with the humerus. x 13.
Fig. 12. Ectoderm plus some mesoderm from the flank of a stage-22 + embryo
grafted into the dorsal surface of the right wing-bud of a stage-22+ embryo. The
length of the humerus is equal to the length of the ulna, x 13.
Effect of limb ectoderm
311
Grafts of flank ectoderm plus mesoderm
Previous experiments had demonstrated that if the dorsal ectoderm plus
some subjacent mesoderm was removed from the dorsal surface of the right
wing and discarded, the right wing developed normally (Stark & Searls, 1974).
It is demonstrated above that limb ectoderm plus subjacent mesoderm other
than dorsal right-wing ectoderm in normal orientation on the dorsal surface
of the right wing may disrupt normal wing development. Experiments were
done to discover what effect non-limb ectoderm plus mesoderm would have
on the development of the right wing. Ectoderm plus subjacent mesoderm
from the flank was grafted into the dorsal surface of the right wing. Of 20 wings
carrying grafts from the flank, only two had ectopic cartilage (Fig. 12) and
none had a short humerus. No attempt was made to control the axes of the
flank grafts before they were implanted, so the implants may have been with
all orientations.
DISCUSSION
Previous experiments have demonstrated that if the ectoderm plus some
subjacent mesoderm is removed from the dorsal surface of the right wing and
not replaced, the wing develops normally (Stark & Searls, 1974); if a graft
fails to take to the graft site the result is the same. Loss of the graft often
may be detected by inspection of the host wing 18 h after the operation, but
it is known that in some cases loss of the graft was not detected by this method.
Some operations of each kind gave rise to normal limbs through failure of
the graft to remain at the graft site. It is possible that grafts moved within
the graft site. It has been demonstrated that if ectoderm on the dorsal surface
of the right wing is moved to a new location on the dorsal surface of the wing
but remains in normal orientation, this will have no effect on normal limb
development (Stark & Searls, 1974). However, rotation of the dorsal ectoderm
does change normal limb development and the graft may rotate within the
graft site. This could not be detected by inspection of the graft 18 h after the
operation. It is also possible that some grafts were made with the axes of the
grafted ectoderm improperly recorded. It is known that one graft was upside
down. From all of these factors, variation is expected in the result, and
considerable variation is observed for each kind of operation.
Grafts in reversed proximal-distal orientation generally caused the humerus
to be short. When grafts were in normal orientation, only 26 % of the wings
had a short humerus. If grafts from the dorsal surfaces of the right and left
legs are neglected, then only 18 % of the wings carrying grafts in normal
proximal-distal orientation had a short humerus. In contrast, 60 % of the
wings carrying a graft in reversed proximal-distal orientation had a short
humerus. If the wings that were normal in every way are neglected (11 out of
76), the other wings all had ectopic cartilage and 73 % of these had a short
378
R. L. SEARLS
humerus. In no case was the effect observed to extend to the radius and ulna;
they were always observed to be the same length and morphology as in the
contralateral control wings. However, wings carrying grafts from the dorsal
surface of the right and left legs in normal orientation tended to have a short
humerus (58 %), particularly wings carrying grafts from the dorsal surface
of the left leg (73 %). Wings carrying grafts from the ventral surface of the
left leg in reversed proximal-distal orientation often had a humerus of normal
length (67 %).
Reversal of the cranial-caudal (anterior-posterior) axis did not seem to
give a definite abnormality. Grafts from the ventral surfaces of the right and
left wings always gave the same sort of abnormalities, but have opposite
cranial-caudal axes. Many other examples can be found in Tables 1 and 2
where grafts with different cranial-caudal axes gave the same limb abnormalities.
Whether the graft be wing or leg seems to be important. Dorsal wing grafts
always gave different results from dorsal leg grafts. Whether the graft be dorsal
or ventral also seems to have some importance; dorsal and ventral grafts
from the same limb usually gave different results. However, grafts from the
dorsal and ventral left wing in normal proximal-distal orientation gave the
same sort of abnormalities, and grafts from the dorsal and ventral right leg
in reversed proximal-distal orientation gave the same sort of abnormalities.
In all of these operations, ectoderm plus a small amount of subjacent
mesoderm was grafted to the dorsal surface of the right wing-bud of a host
embryo. Attempts have been made to reverse the orientation of ectoderm but
not the subjacent mesoderm, but these attempts were unsatisfactory. The
ectoderm curled very rapidly as soon as it became loose from the mesoderm
at room temperature, and was very hard to see by reflected light through the
window in the shell. Thus it was very difficult to be sure of the orientation of
grafted ectoderm. However, it is believed that the grafted mesoderm by itself
would have no effect on normal limb development. Experiments have been
described previously in which blocks of mesoderm alone were grafted into
the right wing-bud (Saunders, Cairns & Gasseling, 1957; Saunders, Gasseling
& Cairns, 1959; Cairns, 1965; Searls, 1967; Searls & Janners, 1969; Stark &
Searls, 1974). In those experiments the mesoderm was obtained from various
regions of the limb, from both wings and legs, and blocks were implanted
that were both larger and smaller than in the present experiments. Ectopic
cartilage was obtained when cartilage-forming cells were implanted in a strange
position in a host wing, but only when the implant was from the cartilageforming region of the limb of an embryo stage 25 or older, or when the whole
elbow region from an embryo stage 23 or 24 was rotated 180°. Ectopic cartilage
was not obtained when the cells were from the limb of an embryo stage 22 or
younger, when the implanted cells were not from the cartilage-forming region,
or when the implant was from the limb of an embryo stage 22-24 but the
implant was small. In the experiments described in this paper, the ectoderm
Effect of limb ectoderm
379
plus subjacent mesoderm was not used if the layer of mesoderm was known
to include cells from the chondrogenic region. In general, the mesoderm
included only cells from the prospective soft tissue region and was grafted
into the prospective soft tissue region. Based on the previous experiments, it
is believed that if the mesoderm were placed under host ectoderm, the mesoderm would regulate.
It cannot be determined from these experiments whether the abnormalities
in the host wings result from some action of the grafted ectoderm alone or
from some interaction between the grafted ectoderm and immediately subjacent
mesoderm. In Ambystoma, rotation of the skin 180° prior to amputation of
a limb produced multiple regenerates (Carlson, 1974). Similar results have
been obtained in other amphibia and in insects (reviewed in Carlson, 1974).
It is difficult to compare the effect of rotation of the ectoderm plus subjacent
mesoderm during limb outgrowth during chick development with the effect of
rotation of the skin during limb regeneration in amphibia, but it is difficult
not to suspect a common mechanism. A number of papers suggest that the
effect of foreign skin on limb regeneration in amphibia may be due to the
dermis rather than the epidermis (see, for example, Glade, 1963). Limb
ectoderm alone can have an influence on normallimb development. A limb
ectodermal jacket (prepared using trypsin to remove some of the material
between the ectoderm and subjacent mesoderm and to loosen the ectoderm)
placed on a limb mesoblast gave distal cartilaginous structures with the dorsoventral orientation of the ectoderm. However, in those experiments the modification of normal limb development was only of the structures that had not
yet been individuated in the mesoblast. In the present experiments a graft
made to the dorsal surface of a stage-24 limb caused modification of the
development of the humerus. By stage 23 the humerus can be easily recognized in
autoradiographs following administration of S-35 sulfate (Searls, 1965; Hinchliffe
& Ede, 1973), and thus might be considered fully individuated prior to stage 24.
The observed modification of normal limb development must result from
an interaction between the graft and the host environment. Ventral right-wing
ectoderm plus its subjacent mesoderm, which can disrupt normal wing
development if grafted to the dorsal surface of the wing, remains in normal
orientation on the ventral surface of the wing after a graft to the dorsal surface
of the wing. If both the dorsal and the ventral ectoderm are reversed 180°, the
result is quite similar to the reversal of dorsal right-wing ectoderm plus
subjacent mesoderm alone (Stark & Searls, 1974); presumably in that case
the dorsal and ventral ectoderm act in concert. In the experiments described
here the ventral ectoderm in normal orientation on the ventral surface must
act antagonistically to the grafted ectoderm, modifying and perhaps in some
cases overcoming the action of the graft.
The effect of the proximal-distal orientation of the graft on the length of
the humerus suggests that the graft may have an influence on the pattern of
380
R. L. SEARLS
growth in the underlying host mesoderm. It may be that the graft causes the
mesoderm cells to accumulate in an abnormal pattern. The cells near the
ectoderm may differentiate as muscle and the remaining cells as cartilage.
Since the distribution of cells is abnormal, the pattern of cartilage would be
abnormal. Cells from the prospective soft tissue region of the limb of a stage-24
embryo are still capable of differentiating as cartilage (Zwilling, 1966).
The experiments described here also supply evidence for a rotation of the
humerus. In many of the limbs containing ectopic cartilage, the humerus
appeared to have rotated 90° in a counter-clockwise direction as viewed from
the tip of the wing. A rotation of the humerus of this kind is not really
surprising. The adult chicken wing (like the human arm) cannot be held with
the bones oriented as they are oriented in the early embryonic limb. After
7 days of embryonic development, the articulation of the radius with the
humerus is cranial to the articulation of the ulna. The radius, ulna and hand
are all on the same plane (Stark & Searls, 1973). After 10-11 days of incubation
the articulation of the radius with the humerus is dorsal to the articulation
of the ulna. Thus, the plane of these articulations has rotated 90° between
7 and 11 days of incubation. In some of the limbs described in this paper the
humerus was short and wide and the articulations of the radius and ulna
could not rotate. In these cases the plane of the wrist and hand was caused
to rotate so that the distal limb came to lie at right angles to the plane in which
they usually lie; rotation of the short and wide humerus caused the whole
of the distal limb to rotate. Rotation of the humerus also explains why ectopic
spurs of cartilage sometimes were attached to the ventral surface of the
humerus (cf. Fig. 4). Presumably the spur formed on the posterior edge of
the humerus during the first 3 days after the operation and then was caused
to rotate to a ventral position as the humerus rotated. At this time I cannot
make any suggestion as to how much of the proximal limb rotates with the
humerus. This problem is under investigation.
Supported by National Institutes of Health Grant HD04669.
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GLADE, R. W. (1963). Effects of tail skin, epidermis, and dermis on limb regeneration in
TrHunts viridescens and Sireden mexicanum. J. exp. Zool. 152, 169-193.
HAMBURGER, F. (1960). A Manual of Experimental Embryology, 2nd ed. Chicago, 111.:
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