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/ . Embryol. exp. Morph. Vol. 44, pp. 15-29, 1978
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The onset of osteogenesis in the developing
chick limb
By NIGEL HOLDER 1
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School, London
SUMMARY
The pattern of the onset of osteogenesis in each of the skeletal elements of the developing
limbs of the chick embryo is described using Alizarin-red-stained whole mounts. Grafting
experiments where 6-day unossified cartilage rudiments are placed into 4-day host wings show
the initiation of ossification to be a programmed event. Truncation experiments show this to
be the case not only for the initial onset of osteogenesis, but also for each region of each
element. These results are discussed in terms of morphogenetic events occurring in the early
wing bud.
INTRODUCTION
In recent years the developing chick wing bud has been extensively used as an
experimental model for studying the main aspects of morphogenesis: pattern
formation, cytodirTerentiation and growth. The work has been mostly concerned with the early stages of limb development, especially with those of
pattern formation (Saunders, 1972; Wolpert Lewis & Summerbell, 1975;
Stocum, 1975). The present investigation was undertaken in an attempt to
elucidate the mechanisms controlling the differentiation of a tissue which does
not appear until pattern formation is complete.
Osteogenesis is first seen in the long bones of the chick wing at stage 32
(Hamburger & Hamilton, 1951), approximately 8 days of incubation. The lightmicroscope histology of this process has been well documented by Fell (1925)
in the chick. In the wing the cartilage anlage of the humerus, radius and ulna
first appear at stage 24 and the proximal distal sequence of limb parts is complete by stage 28 (Summerbell, 1974). The growth of these skeletal elements
follows a characteristic cellular pattern with events always being more advanced
in the diaphyseal regions (Fell, 1925). The shape of the future adult bones is fully
established by stage 36 and the positions of the future articulations are clear
(Holder, 1977). Just prior to the first histological appearance of the diaphyseal
bony collar the adjacent cartilage cells hypertrophy and subsequently die.
The penchondrium, which previously consisted of a layer comprising closely
1
Author's address: Center for Pathobiology, School of Biological Sciences, University of
California, Irvine, California, 92664, U.S.A.
16
N. HOLDER
packed spindle-shaped cells lying at right angles to the flattened cartilage cells,
forms into two layers consisting of an outer fibrous connective tissue region and
an inner osteogenic region made up of newly differentiated osteoblasts, lying
between the perichondrium and the diaphyseal cartilage (Fell, 1925; Jackson,
1957). A thin collar of osteoid is produced by these osteoblasts. It is the control
of this process that is the concern of this study.
Although the histological sequence of these events has been well documented
both in the chick and in man (see Bloom & Fawcett, 1975), the pattern of the
onset of ossification in the chick limb has received little attention, and those
reports that have been published are not in full agreement (see Romanoff, 1960).
However, the pattern of onset is better understood for the human skeleton
(Noback & Robertson, 1951; Gardner, 1971). The first object of this study was,
therefore, to provide an accurate description of the pattern of ossification in the
chick wing and leg. Discrepancies in previous reports have been due largely to
the varying sensitivity of the techniques used to monitor osteogenesis (Noback
& Robertson, 1951). For this study a combination of two techniques - sections
stained with toluidine blue and whole mounts stained with Alizarin red S - was
chosen.
Having accurately established the chronological order of appearance of the
ossification centres in each cartilaginous element of the wing and leg, the next
objective was to establish to what degree this process was programmed both for
the element concerned and for each region of each element. That osteogenesis
is a predetermined process has clearly been shown by Fell (1931), who demonstrated it in explanted chick long bones grown in organ culture. To clarify and
expand on these observations two types of in vivo experiments were designed.
The first involved placing a piece of cartilage from a 6-day long bone (stage 28)
into a wing 2 days younger (stage 24). The implant would normally ossify after a
further 2 days (8 days); but if the onset of ossification was initiated by some
influence outside the cartilage - that is, from some other limb or body tissue then the implant would begin osteogenesis not according to its own programme
but to a new one implemented by the younger experimental host embryo. This
experiment would also show how accurately the programme is expressed. The
second class of experiments involved wings which had been truncated in the
forearm region at various levels at stage 24, 4 days before ossification would
normally begin. These embryos were sacrificed at various times after the operation in order to see if ossification in the regions near the epiphysis occurred at
the expected time, in the absence of any influence from the ossification centres
of the extirpated central diaphyses. This would clarify whether osteogenesis
occurring proximal and distal to the initial central collar was due to programming along the complete length of the cartilage element or to a sequential
induction of bone formation after the initial onset.
Whole skeletal explants from stage-28 wings grown in organ culture were used
to examine further two facets of periosteal ossification that had been revealed by
The onset of osteogenesis in the developing chick limb
17
Fig. 1. Transverse section of a potential graft from a stage-28 humerus. C, cartilage,
P, perichondrium, S, soft tissue x 150.
histological analysis (Fell, 1925): that the cartilage cells adjacent to the initial
site of ossification hypertrophy just prior to the onset of ossification and subsequently die, and that the vascular system plays a prominent role in the process
of ossification.
METHODS
Fertilized White Leghorn embryos were incubated at 38 °C and windowed on
the third day of incubation. The embryos were staged according to Hamburger
& Hamilton (195J) and returned to the incubator until required.
The procedure for preparing whole mounts stained with Alizarin red S was
essentially that of Humason (1962). Embryos between stages 30 (7 days) and
45 (19 days) were fixed in 95 % alcohol for 2 days. The embryos were then transferred to 2 % potassium hydroxide until the skeleton was just visible, approximately 6 h for stage 30-36 embryos, and up to 24 h for stage 45. Embryos were
then immersed in Alizarin red S working solution (Humason, 1962) for 24 h.
Smaller embryos (stages 30-36) were differentiated in 0-5 % and stage-45
embryos in 2 % potassium hydroxide. The specimens were then cleared in varying concentrations of glycerol, and stored in pure glycerol with thymol
preservative. They were examined under a Zeiss binocular microscope.
Embryos from stages 30-36 were fixed in half-strength Karnovsky fixative
18
N. HOLDER
(c)
(b)
A
B
Fig. 2. Diagrammatic representation of the operations performed. (A) Segments of
diaphyseal cartilage were cut out from the humerus, radius or ulna of a stage-28
skeleton and implanted into a stage-24 host, (a) Stage-24 host; (b) stage-28 skeleton;
(c) the completed graft. H, Humerus; R, radius; U, ulna. (B) Stage-25 wings were
cut across at right angles to the proximo-distal axis at various positions relative to
the elbow and wrist, and the distal piece discarded, a, Proximal cut; b, distal cut.
Stippled outline represents the cartilage anlage as indicated by alcian-green-stained
whole-mounts (Summerbell 1976). H, Humerus; R, radius; U, ulna.
The onset of osteogenesis in the developing chick limb
19
Table 1. Completed experiments
Time of sacrifice
after operation
Stage of
implant
Stage of
host
Result
Cases
(A) Diaphyseal pieces from stage-28 humeri, radii or ulnae implanted into
stage-24 wings*
30
26
No ossification
1 day
4
8
2 days
32/33
28
First ossification
of implant
3 days
35
30
Continued ossification 6
of implant
8
4 days
36
32/33
First ossification
of host
Time of sacrifice
after operation
(B) Forearm truncation!
Position of
truncation
33 (8 days)
Mid-forearm
33 (8 days)
39 (13 days)
Proximal-forearm
Mid-forearm
39 (13 days)
Proximal-forearm
Result
Cases
Central diaphyseal
ossification
No ossification
Central diaphyseal
ossification
Proximal diaphyseal
ossification
8
8
10
8
* Grafted wings were monitored for ossification with Alizarin-red-stained whole mounts
and toluidine-blue-stained sections.
f Ossification was monitored with Alizarin-red whole mounts.
(Karnovsky, 1965), dehydrated and embedded in Araldite; 2/mi sections were
cut on a Huxley Cambridge ultramicrotome and stained with toluidine blue.
In vivo experiments
(1) The cartilaginous skeletons from stage-28 wing-buds were dissected free of
soft tissue in a dish of balanced salt solution. The diaphyseal regions of either
the humerus, radius or ulna were excised and transferred to a stage-24 egg,
already prepared for operating. The grafted piece of tissue included the cartilage
of the diaphysis and its intact perichondrium plus small amounts of surrounding
mesenchyme (Fig. 1). A hole, the length of the excised piece of cartilage element,
was cut in the proximo-distal axis of the wing and the implant was placed into
the hole (Fig. 2a). The egg was then resealed and returned to the incubator.
Embryos were sacrificed at various times subsequently (Table 1A), and were
either fixed for sectioning or whole-mounted with Alizarin red S.
(2) Stage-24 or -25 wing-buds were selected and the positions of the elbow and
wrist were marked (Holder, 1977). Cuts were made at various positions relative
to the markers (Fig. 2b), producing truncated limbs with various amounts of
20
(a)
N.HOLDER
1 mm
•n
1 mm
(d)
/
mm
Fig. 3. Significant stages in the pattern of onset of ossification in the wing, as seen
with whole mounts stained with Alizarin red S. (a) Stage 33 (8 days); (6) stage 36
(10 days); (c) stage 39 (13 days); (d) stage 45 (19-20 days).
the forearm region present. Embryos were sacrificed after 8 days (stage 32-33)
and 13 days (stage 39) (Table IB).
In vitro experiments
Stage-28 wing-buds were excised from the embryo and the skeletons dissected free of soft tissue in a dish of balanced salt solution. The skeletons were
cultured on millipore filters (25 jam thick, 0-8 jum pore size) on stainless-steel
grids in Sterilin tissue-culture dishes; 1-5 ml of Biggers' medium (Flow Laboratories) was added to each dish and the medium changed every 3 days. Cultures
were maintained in an atmosphere of 5 % CO2 in air at 38 °C. The pattern of
ossification and the histological appearance of the explants were examined in
sections and whole mounts stained with Alizarin red S.
RESULTS
1. The pattern of osteogenesis
The pattern of the onset of ossification as seen in whole mounts stained with
Alizarin red is shown in Table 2 and Fig. 2>(a-d) for all the skeletal elements of
the wing up to stage 45, just prior to hatching, and in Table 3 and Fig. 4(a-c)
The onset of osteogenesis
in the developing chick limb
21
Table 2. The chronological order of appearance of ossification centres in the
skeletal elements of the developing wing, as shown by Alizarin-red-stained wholemounts
Element
Stage of onset
Days of incubation
Humerus
Radius
Ulna
Digit 2
Metacarpal
Phalanx 1
Phalanx 2
Digit 3
Metacarpal
Phalanx 1
Phalanx 2
Digit 4
Metacarpal
Phalanx 1
Carpal elements
32/33
32/33
32/33
7±/8
7±/8
7^/8
Post-hatching
38
45
—
12
19
33
38
38
8
12
12
33
8
—
—
Post-hatching
Post-hatching
Fig. 4. Significant stages in the pattern of onset of ossification in the leg, as seen with
whole mounts stained with Alizarin red S. (a) Stage 33 (8 days); (b) stage 37 (11 days);
(e) stage 45 (19 days) the foot only. The stumpy first metatarsal is arrowed.
22
N. H O L D E R
1 mm
Fig. 5. Alizarin-red-S-stained whole mount of a stage-34 leg showing the tibia and
fibula. Bone can be seen around the invading blood vessels (arrows) on the tibia
giving a mottled appearance which is contrasted with the smooth appearance of the
fibula. /, Tibia;/, fibula.
D3
T
D4
Fig. 6. Stage-33 wing stained with Alizarin red S showing a grafted piece of cartilage
in the upper arm. H, Humerus; R, radius, U, ulna; D3, digit three metacarpal; D4,
digit four metacarpal. The graft is arrowed.
The onset of osteogenesis in the developing chick limb
23
for all the skeletal elements of the leg. The collar-like appearance of the subperiosteal bone is very striking. In the wing osteogenesis commences at the same
time for the humerus, radius and ulna, stage 32-33. Very soon afterwards (stage
33) the metacarpals of digits 3 and 4 ossify (Fig. 3d), with digit 3 being marginally
ahead. No cases were found where ossification had occurred in the upper arm
and not the forearm; however, the initial ossification was often seen in the upper
arm, forearm and digits simultaneously.
Soon after the initiation of osteogenesis, by stage 34-35, bone could be seen
around the blood vessels which were invading the area (Fig. 5). Circular holes
then appeared in the collar where the blood vessels had broken through and
grown into the lacunae left by the dying chondrocytes of the shaft. As the collar
developed along the shaft towards the epiphyses, grooves in the surface could be
seen as the bone formed around the blood vessels running in the long axis of the
elements.
The initial sequence of ossification in the leg is similar to that of the wing, in
that onset occurs simultaneously in the femur, tibia, fibula and the metatarsals
of digits 2, 3 and 4. Ossification of the phalanges begins sooner in the leg than
in the wing: for example, the first phalanges of digits 2 and 3 have begun ossification in the leg by stage 37, while in the wing the first phalangeal ossification
occurs at stage 39. Two elements of the leg, the fibula and the metatarsal of
digit 1, have only a single epiphysis. The fibula lacks a distal epiphysis and the
spike-like distal tip is covered in a bony collar as early as stage 35 (Fig. 4 b).
Ossification begins in the central region of the element and spreads to the end.
The metatarsal of digit 1 does not exhibit a proximal epiphysis and does not
appear to articulate in any way with the adjacent metatarsal at these early stages.
The distal epiphysis appears similar to those of the remaining three metatarsals
and the short stumpy shaft of the metatarsal of this digit appears to ossify at the
same time as the distal shaft regions of these adjacent elements (Fig. 4).
The carpal elements of the wrist, the phalanx of digit 4, the metacarpal of
digit 2 and the tarsal elements of the ankle do not ossify until after hatching in
the chick. This is similar to the situation in Man, where the carpal and tarsal
elements ossify not only much later than the other limb elements, but at different times relative to each other. The carpal elements also fail to ossify after
100 days in regenerating newt limbs (Smith, Lewis, Crawley & Wolpert, 1974).
Toluidine-blue-stained sections of the different elements of the wing showed
the onset of ossification to be slightly earlier than that seen in the Alizarin-redstained whole mounts. For example, a thin layer of osteoid matrix was visible
in the forearm at stage 32. The short delay may be due to the calcification necessary before the matrix will stain with Alizarin. Experiments using fluorescentlabelled antibodies specific for the collagen type-1 molecule characteristic of
bone matrices have shown that this molecule is present in the inner layers of the
perichondrium of the chick tibia by stage 31 (Von der Mark, Von der Mark &
Gay, 1976).
24
N. HOLDER
Fig. l{a) Stage-33 (8 days) wing truncated at the central forearm level at stage 25
stained with Alizarin red S and whole-mounted, (b) Stage 39 (13 days) wing truncated
at the central forearm level at stage 25 stained with Alizarin red S and whole-mounted,
(c) Stage-39 wing truncated at the proximal forearm level at stage 25 stained with
Alizarin red S and whole-mounted. H, Humerus; R, radius; U, ulna.
2. Grafting and truncation results
The implantation experiments (Fig. 1A) were terminated at various times
after grafting (Table 1 A). After 1 day no ossification was seen in either the host
skeleton or the grafted cartilage. After 2 days the implanted diaphyseal cartilage
began to ossify but the host still showed no signs of osteogenesis. After 4 days
the host elements began ossification in the normal way, by which time the
implants had produced a well-developed collar (Fig. 6). The ossification of the
grafted tissue was usually as well developed as that of the host; however, in
The onset of osteogenesis in the developing chick limb
25
Fig. 8. Transverse section of an ulna cultured for 6 days as part of a whole skeletal
explant from a stage-28 wing. C, Hypertrophied cartilage cells; O, osteoid layer;
P, thin petichondrium. x230.
some grafts the collar appeared to be less complete and thinner, perhaps owing
to trauma or direct surgical damage to the perichondrium during grafting.
The truncation experiments produced wings with various amounts of the
forearm region present after stage 26. After 8 days, only those limbs truncated
at the central diaphysis showed ossification (Fig. Id). Those wings truncated
near to the epiphysis showed no ossification. At 13 days, however, wings truncated in either position showed ossification (Fig. 1 b, c).
3. Organ culture experiments
The onset of ossification was seen histologically in the diaphyseal regions of
all the elements of the skeleton. A thin layer of osteoid matrix appeared around
the periphery of the cartilage and under the thin perichondrium (Fig. 8). This
matrix did not stain clearly with Alizarin red. Ossification did not progress any
further than this initial stage, which was also observed by Fell (1931). On histological examination the normal hypertrophied appearance of the cartilage cells
adjacent to the collar of osteoid was seen. The normal sequence of the onset of
ossification was also seen in the digits of the cultured skeletons (Fig. 9). The
osteoid layer did not develop into the trabecular structure characteristic of
endochondral bone and no breaks were seen in the outer collar.
The histological sequence characteristic of the cartilage cells in vivo also
occurs in vitro. The differential timing of ossification in the metacarpals of
digits 3 and 4 and the first ossification in digit 2 was examined in sections.
26
N. HOLDER
Fig. 9. Transverse section of the digital region of a whole skeletal explant from a
stage-28 wing, cultured for 6 days. A, Digit 3 metacarpal; B, Digit 21st phalanx;
C, hypertrophied cartilage; S, small rounded cartilage cells. Osteoid layer is arrowed.
x92.
Fig. 9 shows the hypertrophied diaphyseal cells of digits 3 and 4 metacarpals and
their bony collars, contrasted with the smaller rounder cartilage cells of the first
phalanx of digit 2 which has not yet reached the advanced hypertrophied stage.
This element subsequently produces the bony collar characteristic of ossification
in vitro.
DISCUSSION
The grafting experiments, where pieces of cartilage from the diaphyseal
regions of the humerus, radius or ulna from stage-28 donor wings were placed
into stage-24 host wings, showed that osteogenesis in these long bones is programmed by this stage. The osteoblasts begin to produce a bone matrix at
specific times, giving rise to a subperiosteal collar of bone in the central diaphyseal regions of the skeletal elements of both the developing wing and leg.
Once ossification has begun, the collar spreads along the shaft towards the
epiphyses which do not ossify until after hatching (Figs. 3, 4). The truncation
experiments clearly show that the osteoblasts along the shaft are programmed
to produce bone matrix at specific times in limb development and that there is
no sequential induction, or wave of competence emanating from the initial
central ossification site. The truncation experiments were performed as early as
stage 24, which is only three stages after the forearm region has been specified
(Summerbell, 1974) and is the stage when the first signs of cartilage differentiation
occur in this region (Searls, 1965; Gould, Day & Wolpert, 1972). They strongly
suggest that osteogenesis is programmed by this early stage in limb development.
The onset of osteogenesis in the developing chick limb
27
Table 3. The chronological order of appearance of ossification centres in the
skeletal elements of the developing leg, as shown by Alizarin-red-stained wholemounts
Element
Stage of onset
Days of incubation
Femur
Tibia
Fibula
Digit 1
Metatarsal
Phalanx 1
Phalanx 2
Digit 2
Metatarsal
Phalanx 1
Phalanx 2
Phalanx 3
Digit 3
Metatarsal
Phalanx 1
Phalanx 2
Phalanx 3
Phalanx 4
Digit 4
Metatarsal
Phalanx 1
Phalanx 2
Phalanx 3
Phalanx 4
Phalanx 5
Tarsal elements
32/33
32/33
32/33
7±/8
7i/8
7i/8
39
38
39
13
12
13
32/33
36/37
7*/8
10*
11
13
37
39
32/33
37
38
38
39
32/33
7*/8
11
12
12
13
38
39
39
39
39
7*/8
12
13
13
13
13
Post-hatching
—
These experiments, however, do not establish exactly when or how the osteoblasts become programmed to begin bone matrix secretion. There seem to be
two possible alternatives. The osteoblasts themselves may be programmed in the
young wing-bud in the same way as are the muscle and cartilage cells and may
subsequently differentiate independently, or they may be induced to begin bone
formation by a localised interaction with the adjacent cartilage cells, once
cartilage differentiation has begun. There is a strong correlation both in vivo and
in vitro between the histological appearance of the cartilage cells and osteogenesis. The onset of ossification may be controlled by some signal passing from the
cartilage cells of each element to the adjacent perichondrial cells to promote the
formation of the inner osteogenic layer (see Lacroix, 1961). This may be likened
to the specific induction of the membrane bones of the skull by certain regions
of the brain (Schowing, 1968). This second alternative would enable the limbs to
28
N. HOLDER
program osteogenesis by an indirect mechanism. If the cartilage cells are
programmed to hypertrophy at a particular time and if hypertrophy provides
the signal for osteoblast differentiation, then osteogenesis would be indirectly
controlled by instructions provided in the early limb-bud (Wolpert, Lewis &
Summerbell, 1975).
The pattern of ossification does not follow a clear proximo-distal sequence.
Similarly in Man, the distal phalanges ossify before the proximal phalanges in
the digits of the hand (Noback & Robertson, 1951). There is no simple correlation between the time of specification of parts in the young wing-bud, which
occurs in a strict proximo-distal sequence, and their subsequent ossification. The
histological sequence of events occurring before, during and after osteogenesis
in, for example, the metacarpals of digits 2 and 3 in the wing, are identical, but
the process occurs at 8 days of incubation in one and after hatching in the other
(Table 2). Thus although the cells in each of these skeletal elements look
identical, they are non-equivalent in the sense used by Lewis & Wolpert (1976).
Two skeletal elements in the leg, the fibula and the metatarsal of digit 1,
appear to be lacking specific parts of the diaphysis and epiphysis (see Fig. 4 and
Table 3). Since the metatarsal, for example, ossifies at the same time as the distal
regions of the adjacent three metatarsals (Table 3), is situated distal to the other
metatarsals and lacks a proximal epiphysis, we may presume that the proximal
region of the element has been lost during evolution and that the element seen in
the present-day chick corresponds to the distal pieces of what was once a complete metatarsal bone. Evolutionary changes of this nature are often found in
vertebrate limbs and have been discussed elsewhere in terms of non-equivalence
(Lewis & Wolpert 1976; Lewis & Holder 1977).
The blood system appears to play an important part in ossification of the long
bones. In vivo the blood vessels seem to break through the initially complete
subperiosteal collar soon after its formation, entering the lacunae created by the
dying cartilage cells of the shaft. The subsequent formation of endochondral
bone and its trabecular structure may be due to the blood vessels ferrying
osteoblasts through the inner bony collar and into the shaft region. This process
does not occur in vitro: the osteoblasts are able to produce the initial collar of
osteoid matrix but ossification proceeds no further because these cells are unable
to enter the shaft. The movement of osteoblasts along the invading blood vessels
can be seen when small collars form round individual vessels, giving a mottled
appearance to some of the elements (Fig. 5). As the bony collar spreads towards
the epiphyses, matrix is laid down in grooves around the now longitudinally
directed blood vessels. It is possible that these grooves later form the Haversian
canals characteristic of the adult bone.
I wish to thank Lewis Wolpert for valuable comment and criticism during the course of
this work, Peter Gould for a critical reading of the manuscript, and the Medical Research
Council for financial support.
The onset of osteogenesis in the developing chick limb-bud
29
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EMB 44