/. Embryol. exp. Morph. Vol. 61, pp. 191-205, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Evidence for a metameric pattern in the
development of the chick humerus
By TRENT D. STEPHENS AND TERESA R. McNULTY
Central Laboratory for Human Embryology, Departments of Pediatrics and
Biological Structure, University of Washington
SUMMARY
It has been proposed that the wing bud is induced by some axial influence at a specific
confined location and that the ZPA is the residual influence of such induction. The purpose
of the present investigation was to test this hypothesis. Tantalum foil barriers were placed
lateral to the mesonephric duct and parallel to the long axis of the embryo in the wing field
of stage-12 to -15 chick embryos. These barriers blocked the somatopleure's communication
with more medial tissues at specific somitic levels. The results of these experiments demonstrate that (1) the limb is not induced at one specific point, (2) portions of the humerus
appear to be induced segmentally along the entire limb field and (3) the ZPA is not induced
by axial structures. We propose a model of wing development suggesting that the humerus
is induced as several separate components which then fuse to form the definitive bone.
INTRODUCTION
The chick wing bud develops adjacent to somites 15-20 beginning at stage 16
(Hamburger & Hamilton, 1951) and there is considerable evidence that the
initial events in wing development are in some way influenced by adjacent axial
structures (Kieny, 1970; Kieny, Mauger & Sengel, 1972). This inductive influence of the axial tissues may be blocked by the imposition of a physical
barrier (Hamburger, 1953) between the somites and lateral plate (MurilloFerrol, 1965; Sweeney & Watterson, 1969).
It has been thought that the inductor may in some way relate to the establishment of the zone of polarizing activity (ZPA) (Balcuns, Gasseling & Saunders,
1970). The ZPA wasfirstdescribed by Saunders and Gasseling (1968) as an area of
mesoderm in the proximal, post-axial portion of the developing wing bud which,
when grafted to the pre-axial border of a wing, induced mirror image duplications of the wing tip. Summerbell (1979) has proposed that the ZPA may be
a self-propagating gradient laid down during early inductive events. Fallon &
Crosby (1977) said of the ZPA, 'possibly only a few cells may determine not
1
Author's address: Department of Pediatrics, RD-20, University of Washington, Seattle,
WA 98195, U.S.A.
7-2
192
T. D. STEPHENS AND T. R. McNULTY
only that a limb bud will grow out at a particular level on the body axis, but
also where the posterior border of that limb will be'.
The suggestion made by Fallon & Crosby (1977) that the wing may be
induced by an influence confined to a site which occupies only a small portion
of the entire limb field is given support by the work of Sweeney & Watterson
(1969). They observed, 'complete absence of the right wing . . . in 10 of 11
specimens in which the anterior edge [of a tantalum foil barrier]... was inserted
at the level of somite 18'. Sweeney & Watterson (1969) thus blocked only the
post-axial half of the wing field but completely inhibited wing development.
Further evidence in support of Fallon and Crosby's (1977) suggestion was obtained by Murillo-Ferrol (1965) and Summerbell (1979). They demonstrated
that the pre-axial half of the wing will not develop when blocked from interaction
with the post-axial half.
The remainder of Murillo-Ferrol's data (1965), however, does not agree with
the idea of a small, post-axial location of the inductive influence. Short barriers
placed in the middle of the limb field resulted in the induction of two limb buds,
one posterior and one anterior to the barrier. This observation suggests that the
limb may be induced from a region that is much broader than that proposed by
Fallon & Crosby (1977).
The purpose of the present study was to repeat and expand upon the observations of Sweeney & Watterson (1969) and to account for the apparent discrepancies between their data and that of Murillo-Ferrol (1965). By doing this
we hoped to explore the extent of inductive influence which establishes the limb
field. The results of these experiments suggest that (1) the limb is not induced at
one specific point, (2) anterior-posterior portions of the wing appear to be
induced segmentally along the entire limb field and (3) the ZPA is not induced
by axial structures. Based upon these points, we have developed a model
suggesting segmental development of the humerus.
MATERIALS AND METHODS
White Leghorn chicken eggs (College Biological Supply Co., Bothwell, WA),
which had been incubated for 60 h at 37-38 °C (stages 12-15; Hamburger &
Hamilton, 1951), were prepared for surgery by removing 1-5-2-0 ml of albumin
and cutting a hole in the side of the egg to allow access to the embryo. A few
drops of sterile saline were placed over the embryo and the vitelline membrane
was removed. The embryos were unstained and the experiments were conducted
with the aid of a Wild M8 Stereomicroscope. The use of this microscope enabled
adequate visualization of the unstained embryos.
The total somites were counted with the first full somite posterior to the otic
placode counted as somite No. 1. A longitudinal slit, four to eight somites long,
was made through the somatopleure at specific somite levels just lateral to the
right mesonephric duct with a finely sharpened tungsten needle. Any embryos
Evidence for a metameric pattern in the chick humerus
193
with severe bleeding were discarded. In 35 control specimens, the eggs were then
sealed with cellophane tape and returned to the incubator. In 248 experimental
embryos, a piece of tantalum foil (0-3-0-8 mm x 1-0-1-5 mm and bent at a 90°
angle lengthwise to give a functional blocking area 0-15-0-4 mm x 1-0-1-5 mm)
was inserted into the slit, thus blocking an average of six somites (range: four to
eight). Only somites adjacent to the limb field were referred to in the results.
Even though the remainder of the barrier blocked more caudal or cranial somites
outside of the wing field, these did not appear to contribute to the observed
abnormality. The eggs were sealed with cellophane tape and returned to the
incubator. In 22 additional experimental embryos, foil barriers were inserted
into the somatopleure perpendicular to the long axis of the embryo.
The embryos were inspected 24 h later and the position of the foil was noted
along with the presence or absence and size of the right wing bud. The eggs were
again sealed with tape and returned to the incubator for an additional 6-7 days.
The chicks were removed from the egg and fixed in 10 % phosphate-buffered
formalin after a total incubation time of 9£ or 10^ days.
The overall morphology of the operated wing and foil location was recorded
and representative specimens were photographed. The length and width of the
stylopod, zygopod and autopod of both the operated wing and the contralateral
unoperated control wing of the experimental and control embryos were measured
and recorded. A drawing was then made of each operated wing with the position
of the foil indicated.
The chicks were decapitated, eviscerated and double stained with toluidine
blue and alizarin red S according to the technique of Burdi and Flecker (1968),
cleared with 1 % KOH and stored in a 2:2:1 mixture of glycerol, ethanol and
benzyl alcohol. Glacial acetic acid (three drops/20 ml) was added to the 50 %
ethanol destaining solution to facilitate the destaining.
The stained chicks were scored for the presence or absence, size and polarity
of each bony component of the wing. Each humerus, both experimental and
contralateral controls, were measured for length, greatest proximal diameter,
greatest distal diameter, and central diameter. The direction of tapering,
proximal or distal, was also noted.
RESULTS
There were 24 of 35 control embryos and 162 of 248 experimental embryos
that survived to 9|-10£ days. One out of the 24 controls had a reduced limb
(the rest were normal) and 31 out of the 162 experimental embryos showed no
wing malformations. There were 131 experimental embryos exhibiting some
degree of limb reduction and 37 of these exhibited no wing on the operated side.
Representative experimental chicks are shown in Fig. 1.
Figures 2 and 3 illustrate some of the general trends in reduction defects
following foil implants. The photographs exhibit stained and cleared experi-
194
T. D. STEPHENS AND T. R. McNULTY
Fig. 1. Nine- and ten-day-old chick embryos following foil barrier implants at
day 2\. Barriers blocking wing somites: (A) 15-17; (B) 15-18; (C) 15-20; (D) 19-20;
(E) 17-20; (F) 16-20.
mental wings with various degrees of reduction. The drawings show the proposed pattern of loss for each photograph as well as the barrier location
resulting in the deficient limb. The pattern of loss in each drawing was based
upon the calculation of proximal, central and distal diameter and overall
length as a percentage of the contralateral control. The pattern of loss in the
remaining wing segments was based upon estimates of the deficient tissue.
Figure 4 illustrates the pattern of bone loss in each reduced humerus. Tables 1
and 2 give the specific data for the figure. Table 1 gives the average dimensions
of the humeri, expressed as a percentage of the contralateral controls and
Table 2 gives the number of reduced humeri in each pattern group (A-F in
Figure 4 and the two tables) resulting from barrier implants at specific somite
levels. The humeri could be divided into six distinct patterns of loss in addition
Evidence for a metameric pattern in the chick humerus
\
!
195
/ . . ' . •
Fig. 2. Patterns of wing skeletal reduction (dotted lines) following specific foil
implants (cross hatching). Left diagrams indicate site of foil implantation. Center
diagrams schematically illustrate the proposed pattern of skeletal reduction based
upon percent of the contralateral control. Photographs at right are examples of limb
reductions following foil implantation at the indicated levels. Foil implant blocking
wing somites: (A) 15-16; (B) 15-18; (C) 15-19.
to a 'normal' group and a group with complete absence of the experimental
humerus.
Figure 4A consists of a group of seven humeri all of which exhibited a decrease in only the central diameter. These humeral reductions resulted from
implants primarily adjacent to wing somites 15-16 (Table 2). Figure 4B consists of the diagrams of 17 humeri exhibiting a decrease in both the central and
proximal regions without decrease in length. These reductions resulted from
implants primarily adjacent to wing somites 15-17 and 15-18 (Table 2). There
is a dramatic change from Fig. 4B to Fig. 4C. The latter consists of a group of
10 humeri all exhibiting a marked reduction of proximal tissue and considerably
reduced in length as compared to the previous groups. These reductions
resulted from implants adjacent to wing somites 15-18 and 15-19 (Table 2).
196
T. D. STEPHENS AND T. R. McNULTY
if i
Fig. 3. Patterns of wing skeletal reduction (dotted lines) following specific foil
implants (cross hatching). Left diagrams indicate site of foil implantation. Center
diagrams schematically illustrate the proposed pattern of skeletal reduction based
upon percent of the contralateral control. Photographs at right are examples of limb
reductions following foil implantations at indicated levels. Foil implant blocking
wing somites: (A) 19-20; (B) 17-20; (C) 16-20.
Figures 4D, E and F exhibit a pattern of loss similar to Figs 4A, B and C,
but approximately reversed in reduction pattern as well as location of implant.
Figure 4D, a group of 17 humeri, exhibits a decrease in the diameter of the
central and distal regions of the bone. These reductions resulted from implants
primarily adjacent to wing somites 17-20. Figure 4E is a rather small group,
six of which resulted from post-axial blocks, and two resulted from pre-axial
blocks. The group exhibited a loss of proximal, central and distal diameter
without a loss in length. There is a dramatic change from Fig. 4E to Fig. 4F,
similar but opposite to that seen between Figs 4B and 4C. Figure 4F, a group
of 14 humeri, resulted primarily from implants adjacent to wing somites 16-20
and 17-20.
In addition to the 150 humeri illustrated in Fig. 4 and listed in Tables 1 and 2,
Evidence for a metameric pattern in the chick humerus
197
Fig. 4. Representations of deficient humeri falling into six basic patterns of loss.
Each humerus was measured for length, proximal diameter, distal diameter and
central diameter. In order to standardize the results, each dimension was expressed
as a percent of the contralateral control humerus and then drawn in relation to a
standard control outline. See Table 1 for specific data for each dimension. The
proximal end of each humerus is indicated by a fragment of the scapula attached to
the humerus.
Table 1. Effects of tantalum foil barrier implants on the humerus*
Pattern of lossf
A
B
C
D
E
F
Complete absence
of wing
Normal wing
No. of
Humeri
7
17
10
17
• 8
14
40
38
Proximal
diameter
Central
diameter
Distal
diameter
94±2
92±1
5O±5
93 ±2
92±2
41 ±3
95±4
62±3
97±4
94±2
86±6
54±5
68 ±5
58±7
64±4
63 ±5
27±9
79±3
62±6
17±6
0
0
0
0
0
98±1
96±2
100 ±2
99±2
Length
0
97±3
62±3
± Standard error.
* Average dimensions of humeri expressed as a percentage of the contraleral controls.
t Patterns of loss (A-F) taken from Fig. 4.
198
T. D. STEPHENS AND T. R. McNULTY
Table 2. Effects of tantalum foil barrier implants on the humerus
Position of foil barrier opposite wing somitest
Pattern of
loss*
A
B
C
D
E
F
Complete
absence of
humerus
Normal
humerus
Totals
Total
humeri 15 15-16 15-17 15-18 15-19 15-20 16-20 17-20 18-20 19-20 20
7
17
10
17
8
14
40
1
_
_
-
4
3
-
1
6
1
-
6
7
1
4
38
5
4
4
3
6 11
12
21
150
12
9
1
_
6
8
7
2
7
8
5
2
1
1
2
2
-
15
26
16
10
10
* Pattern of loss: A-F taken from Fig. 4.
t Only the somites adjacent to the wing field are listed although the barrier usually
extended cranially or caudally for a greater distance.
there were another 12 reduced humeri which were not categorized. One humerus
was broken and could not be accurately measured. In one case the humerus
developed inside the thoracic cavity and was excluded from the remaining data.
One chick exhibited multiple leg and vertebral column malformations perhaps
as a result of membrane interference and the wing was thus excluded from
analysis. One other humerus was markedly shortened but did not exhibit any
taper and thus could not be categorized. In addition to these isolated cases,
there were two groups of humeri which could not be categorized. The first
group, with three members (two with 17-20 blocks and one with a 15-18 block),
resulted in humeri that were reduced in every dimension, length and all three
diameters. Furthermore, all three wings in this group were fused at the elbow.
This latter phenomenon was not seen in any of the other 128 reduced limbs.
The second group, with five members, consisted of humeri with abnormal extra
tissue. Two exhibited proximal spikes, two proximal broadening and one distal
broadening. In addition one humerus with proximal broadening exhibited
a central spike and the other an ectopic bone lying parallel to the humerus. In
the case with the distal broadening, the foil was embedded in the broadened
cartilage. In two of the other four cases, the foil was noted embedded in the
limb bud at the 24 h check, and in one other case the foil was caught in the
membranes and had been moved.
In addition to the above mentioned non-categorized humeri, there were wings
with either normal humeri or absent humeri that should be mentioned. Of the
38 wings with normal humeri, seven were missing distal structures while 31
Evidence for a metameric pattern in the chick humerus
199
Table 3. Effects of tantalum foil barrier implants on the radius, ulna and digits*
Wing somites blockedf 1515-16 15-17 15-18 15-19 15-20 16-20 17-20 18-20 19-20 20
Number of implants
Radius
Reduced
Absent
Total affected
6
12
14
22
14
9
16
29
16
11
8
_
-
7
50
57
_
91
91
7
93
100
_
100
100
19
75
94
34
52
86
13
6
19
9
-
-
0
8
17
25
9
0
Ulna
Reduced
Absent
Total affected
_
-
_
-
_
-
0
0
0
32
18
50
29
43
72
100
100
100
100
7
90
97
63
63
_
27
27
_
38
38
Digit II
Reduced
Absent
Total affected
—
-
8
-
_
93
93
6
8
_
73
73
100
-
0
_
29
29
88
94
79
79
25
25
27
27
0
Digit III
Reduced
Absent
Total affected
_
-
—
-
18
41
0
59
7
71
78
100
-
0
_
12
12
100
100
100
7
79
86
6
31
37
36
36
13
13
26
0
0
-
23
27
50
21
57
78
100
100
100
100
3
97
100
6
63
69
27
27
38
38
Digit IV
Reduced
Absent
Total affected
7
7
100
* Each number is represented as a percent of the total implants at that level.
t Only the somites adjacent to the wing field are listed although the: barrier usually
extended cranially or caudal ly for a. greater distance.
were normal wings. Two pre-axial blocks (15-17 and 15-18) resulted in loss of
the radius and reduction of the digits while retaining a normal humerus. Five
post-axial blocks (17-20, 18-20(2), 19-20, 20) resulted in loss of the ulna and
usually the fourth digit in the presence of a normal humerus. On the other hand,
of the 40 wings with no humerus, three exhibited growth of distal tissue, a wing
tip consisting of part of the ulna or radius and one or two reduced digits. All
three of these latter wings resulted from barriers adjacent to wing somites 15-19.
Table 3 demonstrates the effect of the position of foil implants on the other
components of the wing. The radius appeared to be most strongly affected by
barriers positioned adjacent to somites 15-18, 15-19, 15-20 and 16-20. The
ulna appeared to be most strongly affected by barriers positioned adjacent to
somites 15-20, 16-20 and 17-20. Digit II appeared to be strongly affected by
barriers positioned adjacent to somites 15-19, 15-20, 16-20; digit III appeared
to be affected primarily by barriers positioned at levels 15-20 and 16-20.
Digit IV appeared to be most strongly affected by barriers positioned opposite
wing somites 15-20, 16-20 and 17-20.
200
T. D. STEPHENS AND T. R. McNULTY
B
Fig. 5. (A) Ten-day-old chick embryo with two right wings resulting from a foil
barrier implanted perpendicular to the long axis of the embryo adjacent to somite 18.
(B) Right wings of the chick embryo stained with alizarin red and toluidine blue
shown in (A). The pre-axial reduced wing (a) exhibits only a proximal portion
of the humerus. The post-axial reduced wing exhibits a distal portion of the
humerus (b), the ulna and digits III and IV.
Figure 5 illustrates the result of a foil barrier implanted perpendicular to the
long axis of the embryo. Figure 5B depicts the stained specimen with the
humerus divided into a proximal, pre-axial portion and a distal, post-axial
portion. Fifteen of 22 embryos with the barrier placed perpendicular to the long
axis of the embryo survived the surgery. Eleven embryos exhibited split humeri
(Fig. 5) and the remaining four exhibited multiple tissue nodules.
The limb polarity was considered abnormal if any structure which developed
Evidence for a metameric pattern in the chick humerus
201
distal to the elbow was abnormal in its positioning or orientation. Of 94 malformed wings exhibiting growth beyond the elbow, 11 were found to have some
degree of abnormal polarity. Only one of the 'abnormal' limbs exhibited what
might be considered mispositioning of the digits consisting of what appeared to
be two number-IV digits on the same wing. The remaining ten consisted of
wings in which the elbow was bent in an abnormal direction (cf. Fig. 3B). Six
of the 11 resulted from post-axial blocks (i.e. somites 16-20, 17-20 or 18-20)
and five resulted from pre-axial blocks (i.e. somites 15-19 or 15-18).
DISCUSSION
One purpose of the present study was to repeat and expand upon the observations of Sweeney & Watterson (1969) and to account for the apparent discrepancies between their data and that of Murillo-Ferrol (1965). Our results
suggest that the limb is induced at several positions along its anterior-posterior
length and not at one small post-axial location. This data contradicts the data
of Sweeney & Watterson (1969) which led to the suggestion of a post-axial
induction of the entire wing, but supports the observations of Murillo-Ferrol
(1965) suggesting a broader influence.
Another purpose of the present study was to test the hypothesis that the ZPA
is a post-axial residue of the initial induction of the limb. Our data suggest that
the ZPA is the source of an influence which is independent of any axial induction
of the limb. We present here two pieces of evidence that the ZPA is not induced
by axial structures, at least after stage 12 of development. First, of 94 wings
exhibiting growth beyond the elbow, only 11 were found to have some degree of
abnormal polarity. Of the 11, 6 resulted from post-axial blocks and 5 resulted
from pre-axial blocks. Therefore, barriers separating the post-axial limb field
(ZPA) from the somites had no more effect on polarity than barriers separating
the pre-axial (non-ZPA) region of the limb field from the somites. Second,
barriers blocking somites 18-20 resulted in limbs with a portion of the humerus
radius and often digits II and III present (Fig. 6 A). On the other hand, barriers
placed perpendicular to the long axis of embryos between somites 17 and 18
resulted in two partial limbs (Fig. 6B). The partial limb post-axial to the barrier
developed a partial humerus, an ulna and digits III and IV. The partial limb
pre-axial to the barrier developed only a portion of the humerus. Therefore,
a block parallel to the long axis of the embryo appears to allow more distal
pattern formation in pre-axial tissue than a block perpendicular to the long
axis at the same level. This would suggest that with a parallel barrier (Fig. 6 A),
a post-axial influence (ZPA) is not inhibited and can facilitate development of
pre-axial structures. On the other hand, a perpendicular barrier (Fig. 6B) blocks
the post-axial influence from reaching the pre-axial tissue and thus prevents the
ZPA from facilitating the subsequent distal development of pre-axial structures.
Three pieces of data suggest that the proximal and distal humeral segments
202
T. D. STEPHENS AND T. R. M c N U L T Y
ZPA
ZPA
Fig. 6. Schematic representation of the influence of the ZPA upon wing pattern
formation. (A) Illustrates the effect of placing a barrier between the presumptive
ZPA and the adjacent wing somites (18-20). (B) Illustrates the effect of placing
a barrier between the presumptive ZPA and the pre-axial limb tissue perpendicular
to the long axis of the chick between somites 17 and 18.
described above exhibit a pre-axial and post-axial tapering, respectively (cf.
Figs 2, 3 and 7). First, it may be noted that the two humeral segments resulting
from a perpendicular foil implant consisted of entirely different regions of the
bone (Fig. 5B). The humeral segment which developed pre-axial to the barrier
was the proximal head of the bone with a gradual distal tapering. The partial
humerus which developed post-axial to the perpendicular barrier consisted of
the distal end of the bone and a gradual proximal tapering. Second, the trend
Evidence for a metameric pattern in the chick humerus
203
Fig. 7. (A) The proposed pattern of humeral components based upon observations
of foil barrier induced limb deficiencies. The double hatched area was obtained as
a mean from loss pattern Fig. 4 A (showing tissue lost) and assumed to be pre-axial
because of the pre-axial locations of the foil producing the group. The white area
was obtained as a mean from loss pattern Fig. 4F and the stippled, area from
Fig. 4C (both showing tissue present). The tapering direction, post-axial or preaxial, in the white and stippled areas was assumed based upon post-axial or preaxial location of implants and also upon Figure 5B (see discussion). (B) Schematic
representation of 'sclerotomal' patterns of referred pain from a human humerus
(After Inman & Saunders, 1944).
of reduction in Fig. 4 was proximal loss associated with pre-axial barriers
(Fig. 4 A, B and C) and distal loss associated with post-axial barriers (Fig. 4D,
E, F). Third, the radius was more often missing in association with loss patterns
A, B and C, while the ulna was seen to be lost with loss patterns D, E and F.
These observations of humeral reduction trends have led us to postulate
a model of humeral development. According to this model, the humerus
develops, not as a single bone, but as a mosaic of several components which in
some way unite to form the complete bone (Fig. 7 A). These components are in
turn induced, or at least influenced, by axial or para-axial structures in a segmental fashion. The model does not propose that this influence emanates
specifically from the somites (indeed, Chevallier, 1978 has demonstrated that
removal of somite mesoderm does not interfere with normal wing skeletal
patterning), but rather that the foil barrier blocks the lateral migration of an
influence of unknown identity. Hence, the model proposes the existence of some
influence that stimulates the development of individual humeral segments which
together form a complete bone.
The fact that a barrier placed perpendicular to the embryo axis results in the
204
T. D. STEPHENS AND T. R. McNULTY
development of two complementary humeral components lends strong support
to such a model. Furthermore, partial or split humeri were reported by MurilloFerrol (1965) and can be seen in the photographs of Summerbell (1979). Inman
& Saunders (1944) demonstrated patterns of referred pain from the human
humerus that are strikingly similar to the pattern of loss which we have observed
in the chick humerus (Fig. 7B). In addition, one of the major models of limb
evolution proposes that metameric basals at some point in phylogeny fused to
form the primitive humerus (cf. Jarvik, 1965). These observations all strongly
suggest the development of a mosaic humerus.
In conclusion, we have demonstrated that the development of the limb
skeletal pattern may be altered by the imposition of a barrier medial to the limb
field. The results of such alterations have led us to conclude that the humerus,
and to some extent distal limb components, develop by the united action of
several sub-components which together make up the definitive bone. The
precise time of action and nature of the axial influence upon such a pattern will
be the subject of future investigation.
This research was partially funded by NICHD grant HDOO836.
We are particularly grateful to Drs Thomas Shepard and Alan Fantel for their assistance
and to Drs Ray L. Waterson and John W. Saunders, Jr for technical advice. We are also
grateful to Barbara Brownfield for help with preparation of the manuscript.
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{Received 8 April 1980, revised 12 August 1980)