/ . Embryo!, exp. Morph. Vol. 68, pp. 1-7, 1982
Printed in Great Britain © Company of Biologists Limited 1982
The proximodistal determination of skeletal parts
in the developing chick leg
By DONENE A. ROWE AND JOHN F. FALLON 1
From the Department of Anatomy,
The University of Wisconsin
SUMMARY
Currently the chick leg bud or its components are being used extensively to study questions
in development. Although fate maps of the leg, similar to that developed by Saufiders
(1948) for the wing, have been available (Hampe, 1957 a, 1959), no study of the proximodistal
sequence of the specification of leg structures exists. Such a sequence developed fot the
wing by removal of the apical ectodermal ridge at successive stages (Saunders, 1948; Surrjmerbell, 1974), has proven useful to the study of wing development. In this paper, we present
a similar proximodistal sequence for the chick leg including the same developmental stage
range as that of the wing sequence.
INTRODUCTION
The chick wing bud has been used extensively as a model for amniote limb
morphogenesis (reviewed in Zwilling, 1961; Saunders, 1977). In addition, legbud components used in combination with those of the wing have been useful,
for in the resulting composite limbs, leg and wing structures can be distinguished
from one another (Kieny, 1964; Pautou & Kieny, 1973). Many similarities
exist between the two limb buds. For example, the presence of a polarizing
region along the posterior border (Saunders, 1972) and the requirement for
a period of association with the somites for normal development (Kieny, 1969,
1970). Moreover, leg-bud ectoderm allows outgrowth of wing structures When
combined with wing-bud mesoderm and vice versa (Zwilling, 1956, 1964;
Saunders, Cairns & Gasseling, 1957). However, differences between leg and
wing have been noted, including the timing of events leading to normal
morphogenesis (Kieny, 1968, 1969, 1970) and the gross morphology of the
leg and wing buds. This is particularly the case with the apical ectodermal
ridge which, in the avian wing bud, is uniquely asymmetrical, thicker posteriorly
than anteriorly (Zwilling, 1961). In contrast, the avian leg and the limb buds
of other amniotes possess a ridge of symmetrical thickness anteriorly and
1
Author's address: Department of Anatomy, The University of Wisconsin, Ma<Jison,
Wisconsin 53706, U.S.A.
2
D. A. ROWE AND J. F. FALLON
posteriorly. Whether morphological differences reflect any mechanistic differences in the morphogenesis of the wing and leg is unknown.
Hampe (1956a, 1957a, 1959) has provided a map which shows the placement
of the precursor cells of leg structures. This map, based upon experiments
utilizing carbon markings and the development of isolated sections of the leg
bud, allows localization in the bud of structures already determined, proximal
to the proposed progress zone (Summerbell, Lewis & Wolpert, 1973), at the
stages used for experimentation. However, the results of Hampe's experiments
do not demonstrate the most distal structures determined at successive stages
of development, as has been done in the wing by apical ridge removal (Saunders,
1948; Summerbell, 1974). Considering the increasing interest in the use of the
leg as an experimental system, a sequence similar to that for the wing is
necessary as a common reference. Therefore, in this paper, we present a proximo-
distal sequence of determined elements for the leg at stages 17-29.
MATERIALS AND METHODS
Fertilized White Leghorn eggs were incubated at 38 °C for 2f-3 days. At
that time they were candled and fenestrated according to the technique of
Zwilling (1959). The stage of the embryo was determined by somite number at
stage 17, by the ratio of the length to the width of the wing and leg buds for
stages 18-21, and by other criteria as described by Hamburger & Hamilton (1951)
for all stages. At each of the stages 17-29, the apical ectodermal ridge was
removed from the right leg bud with a fine glass needle. The eggs of operated
embryos were then sealed with Scotch tape and returned to the incubator.
Embryos were checked at 24 h and those with incomplete ridge removals were
discarded. A ridge may form after removal of the apical epithelium from stage-17
and-18 leg buds (Kieny, 1968; Fraser & Abbott, 1971) and such ridge formation could be distinguished from cases of incomplete ridge removal. Incomplete
removal resulted in an irregularly shaped limb bud, while those legs with a
complete ridge, which formed after healing of the epithelium, had a normal
limb contour. The few embryos which exhibited normal ridges after stage-17
to -18 removal were allowed to develop with the embryos from which the
ridge was completely removed. The embryos were allowed to develop until
the 10-12 day of incubation. They were then fixed in 10 % formalin and stained
with Victoria blue and cleared.
RESULTS
After the apical ectodermal ridge was removed from the leg bud, a truncated
limb resulted. As the age of the embryo at the time of operation increased, the
resulting legs became more complete distally. Typical examples of the legs
which developed after apical ridge removal and a diagram summarizing the
results appear in Figs. 1-11.
Proximodistal development of the chick leg
i
Fig. 1. The leg elements resulting from removal of apical ridge at stage 18: the
femur and head of the tibia/fibula.
Fig. 2. The leg elements resulting from removal of apical ridge at stage 19: the
femur and about half of the tibia/fibula.
'
Fig. 3. The leg elements resulting from removal of apical ridge at stage 20: the
femur and most of the tibia/fibula.
Fig. 4. The leg elements resulting from the removal of apical ridge at stages 21
and 22: the femur, tibia/fibula, and proximal tarsals fused to the tibia (arrow).
Fig. 5. The leg elements resulting from removal of apical ridge at stage 23: the
femur, tibia/fibula, proximal (arrow) and distal tarsals (arrow head).
Fig. 6. The leg elements resulting from removal of apical ridge at stage 24: the
femur, tibia/fibula, proximal tarsals fused to the tibia (arrow) and distal tarsal
fused to the metatarsals (arrow head), and the proximal ends of the three leg*
metatarsals.
i
D. A. ROWE AND J. F. FALLON
8
10
Fig. 7. The leg elements resulting from removal of apical ridge at stage 25: the
femur, tibia/fibula, tarsals and metatarsals. From this lateral view, it is difficult
to distinguish each of the three metatarsals. In the limb in this photograph, each
of the metatarsals has developed completely.
Fig. 8. The leg elements resulting from removal of apical ridge at stage 26: the
femur, tibia/fibula, tarsals, metatarsals, and proximal phalanges.
Fig. 9. The leg elements resulting from removal of apical ridge at stage 27: the
femur, tibia/fibula, proximal tarsals (arrow), distal tarsal (arrow head), fused
with the metatarsals, and phalangeal elements.
Fig. 10. The leg elements resulting from removal of apical ridge at stage 28: the
femur, tibia/fibula, tarsals, metatarsals, and phalangeal elements.
At stages 17-19, a morphologically identifiable apical ridge had not yet
formed in the leg. When the apical epithelium was removed from stage-17 and
-18 leg buds, normal limbs resulted in a small number of cases, three often at
stage 17, and three of twelve at stage 18. In the remainder of cases, embryos
from which the apical epithelium was removed at stage 17 developed legs with
pelvic girdles and incomplete femurs. In the majority of cases, a femur approximately 90 % of the length of the embryos left femur was the most distal element
formed. Nine of twelve operations performed on stage-18 embryos yielded
legs which were truncated at a level just distal to the condylar end of the tibia
and head of the fibula (Fig. 1). When the apical epithelium had been removed
Proximodistal development of the chick leg
Fig. 11. A drawing of the chick leg. The lines intersect the leg at the approximate
level at which the leg is truncated after removal of apical ridge at the stages
indicated. On the left is the pelvic girdle to which is attached the femur. Articulating
with the femur are the tibia and fibula, with the smaller fibula lateral to the tibia.
Fused to the distal end of the tibia are the proximal tarsals; these fused structures
comprise the tibiotarsus. Distal to the joint is the distal tarsal which is fused with
the metatarsals 2, 3, and 4, comprising the tarsometatarsals. Thefirstdigit originates
from the plantar surface articulating with the very small first metatarsal. Digit 2
articulates with metatarsal 2 and has three phalangeal elements, digit 3 articulates
with metatarsal 3 and has 4 phalangeal elements and digit 4 articulates with metatarsal 4 and has 5 phalangeal elements.
at stage 19 (Fig. 2), all embryos developed legs with the distal-most element
consisting of about half of the tibia/fibula. In some of these cases, the fibula
failed to develop.
By stage 20, removal of the ridge (ten cases) resulted in a range of outgrowth
of the tibia from a level just slightly distal to half of its normal length to a level
about three-quarters of its normal length (Fig. 3). Legs resulting from ridge
removals at stages 21 (twelve cases) and 22 (ten cases) (Fig. 4) were very similar
and extended distally to a level just distal to the proximal tarsals (which are
fused with the tibia to make up the tibiotarsus), while those from removals at
stage 23, 15 cases (Fig. 5) included the distal tarsal (which, in the normal
leg, is fused with the metatarsals to make up the tarsometatarsals). Embryos
from which ridge was removed at stages 24 (14 cases) and 25 (10 cases) developed
increasing lengths of the three major metatarsals of the leg (Figs. 6, 7).
Between stages 26 and 29 (30 cases), removal of ridge resulted in elimination
of decreasing numbers of phalangeal elements (Figs. 8-11) until at stags 29,
removal affected only the distal-most phalanges.
,
DISCUSSION
In this paper, we present a proximodistal sequence of the determination of
skeletal elements for the chick leg during stages 17-29. Legs resulting from
stage-17 and -18 apical epithelium removal were normal in a low percentage
6
D. A. ROWE AND J. F. FALLON
of cases, comparable to results shown by other investigators (Kieny, 1968;
Fraser & Abbott, 1971). It is possible that the conditions for induction of a
functional ridge in the healed epithelium remain in the mesoderm of a very
few stage-17 and -18 leg buds (Kieny, 1968). The remainder of removals from
stage 17 and 18 and removals at all other stages demonstrated the proximodistal
sequence of the determination of leg structures at stages 17-29.
In an extensive series of experiments, Hampe (1956a-c, 1957a, b, 1958, 1959,
1960) developed fate maps for the structures in the mesoderm of the leg through
stage 23. Hampe's fate maps (1957a, 1959) do allow prediction of which
structures have been determined proximally and where their precursors exist
in the mesoderm. In contrast, the representation of the proximodistal sequence
of development presented here shows the most distal structures laid down
during leg bud stages 17-29. Comparable representations which have been
developed for the wing (Saunders, 1948; Summerbell, 1974) have proven useful
to investigators using the wing as an experimental model. Summerbell (1974)
has shown that in the wing the specification of carpal elements spans four
stages. It is interesting to note that this time requirement is similar for the
tarsals and proximal metatarsals of the leg which are specified during stages
21-24. Further experiments are required to explain why this area takes longer
to be specified than the rudiment of a long bone.
With the increasing use of the leg for experimentation, a proximodistal
sequence of the leg will be required for analysis of results and can be used as
that of the wing has been. The combination of Hampe's fate maps and the
proximodistal sequence of determination presenter! here will be a useful tool
for analysis of future experiments on the leg.
These studies were supported by NSF grant no. PCM7903980 and NIH grant no
T32HD7118. We are grateful to Drs Allen W. Clark, Edward Schultz, David B. Slautterback
and Ms. Eugenie Boutin for their careful reading and constructive criticism of the manuscript.
We thank Ms. B. Kay Simandl for technical assistance, Ms. Lucy Taylor for the drawing,
and Ms. Sue Leonard for typing the manuscript.
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{Received 14 August 1981, revised 13 October 1981)