/. Embryol. exp. Morph. Vol. 19, 3, pp. 327-39, May 1968
With 4 plates
Printed in Great Britain
327
Abnormalities in bone and cartilage development
in the talpid3 mutant of the fowl
By J. R. HINCHLIFFE 1 & D. A. EDE2
From the Department of Biological Sciences, Wye College (University of
London); the Department of Zoology, University College of Wales,
Aberystwyth; and the Agricultural Research Council Poultry
Research Centre, Edinburgh
The complex pleiotropic pattern of damage caused in chick embryos by the
talpid3 gene has been described previously by Ede & Kelly (1964#, b) and
Hinchliffe & Ede (1967). The pattern of segregation of the mesenchyme in
forming the precartilaginous or membranous skeleton is abnormal, and the
resulting cartilaginous skeleton shows characteristic fusions of the vertebrae and
of the limb elements. By 11 days of development there is a complete failure of
cartilage-replacement bone to appear, even in the more normally formed cartilage rudiments (e.g. ribs, scapula, coracoid, ilium). By contrast, ossification
to give the membrane bones (the clavicle and bones of the skull and jaws),
which are formed directly from condensations in the mesenchyme, proceeds
normally. This paper describes the attempts made by experimental and
histochemical means to account for the failure of cartilage-replacement bone
formation.
The shoulder-girdle region was chosen for detailed study, as it has the advantage of consisting of a combination of a membrane bone (clavicle) and
cartilage-replacement bones (coracoid and scapula). Chorio-allantoic grafting of
the prospective talpid3 shoulder girdle region was carried out for two reasons:
(a) to obtain an adequate supply of talpid3 shoulder-girdle material, as most
talpid3 embryos die at an early stage, and (b) to determine whether the origin
of the abnormality is humoral or autonomous.
METHODS
r
Living donor embryos were identified by the shape of the wingbuds as normal
or talpid3 at 4 | days, and removed from the egg under sterile conditions to chick
Ringer's solution. The forelimb bud was cut off, leaving a stump representing
1
Author's address: Department of Zoology, University College of Wales, Penglais,
Aberystwyth, Cardiganshire, U.K.
2
Author's address: Poultry Research Centre, West Mains Road, Edinburgh 9, U.K.
328
J. R. HINCHLIFFE & D. A. EDE
the base of the presumptive humerus. A rectangle of body wall measuring
about 2 x II mm, with the stump in the centre, was excised (see Text-fig. 1), then
grafted to the chorio-allantois of an 8-10 day host egg (Hamburger, 1960), and
harvested after a specified period of further incubation. The resulting graft was
dissected in chick Ringer's solution, skin or feathers removed, and a drawing
made of the main bones and cartilages before fixing.
Gross morphology was established by staining whole grafts to make bone or
cartilage clearance preparations. The alizarin-red method was used to show
ossification (Cowdry, 1952) and Van Wijhe's methylene blue method (Cowdry,
1952) for cartilage. A few grafts were stained for both bone and cartilage by the
alizarin red/toluidine blue method of Walley-Wiliams (1941).
Text-fig. 1. Prospective shoulder-girdle region at 4£ days excised for chorio-allantoic
grafting.
For alkaline phosphatase the Gomori (1952) technique was used. Slides were
incubated for 2 h in the incubating medium. Decalcification of the grafts was
not attempted, as this renders impossible the Gomori technique, and as calcification had rarely proceeded far enough to make sectioning difficult. Every third
or fourth slide was stained for alkaline phosphatase. Control slides, treated
identically to the others, but with the omission of the substrate from the incubating medium, enabled deposits of cobalt sulphide due to the presence of calcium
apatite in bone tissue to be distinguished from deposits of calcium phosphate
formed by alkaline phosphatase activity.
The remaining slides were treated in a variety of ways. The Kossa silver
nitrate method (Gomori, 1952) for calcium deposits was used to indicate
mineralization by apatite of the body tissue. Slides were stained for acid or neutral
mucopolysaccharides by the Trevan modification of the PAS technique, fully
described in our previous paper (Hinchliffe & Ede, 1967). The Trevan method,
applied to pairs of slides, one of which had previously been incubated for 1 h in
1 % diastase in distilled water at 38 °C, was also used to stain glycogen. Slides
were stained with toluidine blue to test the metachromatic reaction of talpid3
cartilage.
Talpid3 abnormalities
329
For general morphology, slides were stained in alcian blue and then, after
fixing the stain in borax-saturated alcohol as in the Trevan technique, in Mayer's
acid haemalum as a nuclear stain. Other slides were stained by haematoxylin and
eosin, and by alcian blue/chlorantine fast red (Lison, 1954).
The numbers of grafts obtained at various ages and the method of treatment
are given in Table 1.
Table 1. Treatment of grafts
Total age
(days)
Days in
graft
Sectioned and
stained for
Transparency
alkaline
Blocked in
phosphatase
wax
Cartilage
Bone
Carnoy
fixation
Talpid3
9
11
13-16
41
61
81-111
4
3
10
1
—
3
4
3
1
1
8
2
4
11
Normal
9
11
13-16
41
61
3
2
4
—
2
8
3
Totals: Talpid , 29 grafts; normal,45 grafts.
RESULTS
3
Gross structure of talpid and normal shoulder-girdle grafts
Normal shoulder girdle grafts of total age 13-16 days (grown for 8-11 days
on the chorio-allantois) reveal clearly recognizable elements of the shoulder
girdle (Text-fig. 2). The clavicle can be found in most of the grafts (30 out of
37) and it takes the form of an opaque white splinter of bone, thickened ventrally. The coracoid is still largely cartilaginous, with a central bony collar, and
frequently makes contact ventrally with the cartilaginous sternum. Bone replacement of the curved scapula is further advanced. In addition to the shouldergirdle elements, the head and the ossifying shaft of the humerus are generally
recognizable. Ossification occurs in the grafts at the same time as if the shouldergirdle region had been left in situ in the donor embryo, though the grafted
shoulder-girdle elements are considerably smaller.
Talpid3 shoulder-girdle grafts grown for about 10 days differentiate according
to their prospective genetic fate. The clavicle can be found in most of the
grafts (18 out of 26) and it takes the form of a thin white bone splinter thickened
ventrally, and indistinguishable from the clavicle in normal grafts. The two
cartilage replacement 'bones' of the shoulder girdle, the coracoid and scapula,
show no sign of ossification and are present only as cartilages (see Text-fig. 2).
The humerus also is present only as a cartilage. One alizarin clearance preparation is, however, exceptional in showing a few fragments of disorganized ' bone'
330
J. R. HINCHLIFFE & D. A. EDE
tissue positive for alizarin red adhering to the humerus. The absence of replacement bone in the grafted coracoid and scapula is also a characteristic of their
counterparts in situ in the living 11-day talpid3 embryos (Ede & Kelly, 19646).
The talpid3 cartilage models of the shoulder girdle differ from those of the
normal in other respects: a greater proportion of the talpid3 grafts fail to differentiate identifiable elements, which in some of the poorly differentiated
grafts consist of a confusing and tangled mass of cartilage quite different from
even poorly differentiated normal grafts. Even in the better differentiated grafts
the talpid3 cartilage models are frequently distorted.
Text-fig. 2. Drawings of well-differentiated normal (c and d) and talpid* {a and b)
left shoulder-girdle grafts (from photographs of bone transparencies). Grown on
chorio-allantois: a, for 9 days; b, for 11 days and c and d, for 10 days. Heavy
stippling: membrane bone of clavicle. Light stippling: cartilage-replacement bone.
Cl, Clavicle; Co, coracid; H, humerus; S, scapula.
Histological and histochemical studies
3
Normal and talpid membrane bone {clavicle). The clavicle in both normal and
talpid3 grafts can first be identified at 9 days. In striking contrast to the talpid3
cartilage-replacement bone the talpid3 clavicle develops in the same way as
Talpid3 abnormalities
331
3
the normal (Plate 1, compare fig. B with fig. A). Talpid osteoblasts, rich in
alkaline phosphatase (plate 1, fig. D) and with eccentric nuclei and basophilic
cytoplasm, resemble normal osteoblasts. By 11 days the clavicle develops into a
solid splinter of bone composed microscopically of osteocytes embedded in
lamellae of mineralized osseous material positive for neutral mucopolysaccharides. At a later stage of development, sections of the talpid3 clavicle
indicate its histological and histochemical normality (Plate 4, figs. W, Y).
Normal cartilage-replacement bones. The crucial sequence of events in the
replacement of cartilage by bone (described in detail by Fell, 1925) is shown
by the changes in coracoid and scapula of 9 and 11 days in grafts. Development
takes place earlier in the scapula than in the coracoid. Initially the cartilage rudiments are present without any sign of bone formation, with 'young' chondroblasts possessing an elongated densely staining nucleus and having their long
axis roughly at right angles to the long axis of the rudiment (Plate 1, figs. A, E,
G). At the centre of these rudiments enlargement of the cartilage cells typical of
hypertrophy is beginning, accompanied by the loss of the initial orientation of
the chondroblasts and by the appearance in the cytoplasm of glycogen, which is
typical of hypertrophic cartilage (Henrichsen, 1958). During these two stages
there is almost no alkaline phosphatase activity in the rudiments (Plate 1,
fig. C; Plate 2, fig. I).
The first sign of the differentiation of the perichondrium into a periosteum is
the simultaneous appearance of alkaline phosphatase activity (Plate 1, fig. C)
and cytoplasmic basophilia in the inner layer of closely packed mesenchymal
cells (Plate 2, fig. K). This stage is found in the 9-day coracoid. At the next
developmental stage the perichondrium has differentiated into a periosteum,
with an inner osteogenic layer of osteoblasts characterized by oval nuclei, basophilic cytoplasm, dense packing (Plate 2, fig. M) and by intense alkaline phosphatase activity (Plate 2, fig. I). Alkaline phosphatase-rich osteoblasts differentiate in the periosteum before the cartilage has reached the stage of hypertrophy
at which alkaline phosphatase appears.
Later, hypertrophy of the cartilage reaches its final stage (e.g. the 11-day
scapula: Plate 3, fig. Q), in which alkaline phosphatase makes its appearance in
both the chondroblasts and the matrix. The chondroblasts are degenerating, as
is shown by the loss of chromatin material from the irregularly shaped nucleus
and by vacuolation of the cytoplasm. Mesenchymal cells are eroding the
cartilage rudiment which is surrounded by bone (Plate 3, fig. S). The inner
osteogenic layer of the periosteum consists of a layer of osteoblasts intensely
positive for alkaline phosphatase, overlying the bony matrix containing osteocytes which are devoid of the enzyme. The bony matrix is positive for neutral
mucopolysaccharide and is mineralized.
In later stages—for example, in the 14-day coracoid (Plate 4, figs. V, X)—the
whole central core of the cartilage rudiment becomes hypertrophic and intensely
positive for alkaline phosphatase. Considerable erosion of the rudiment has taken
22
JEEM 19
332
J. R. HINCHLIFFE & D. A. EDE
place, and substantial amounts of bone have now been laid down in a collar
round the diaphysis of the cartilage.
Talpid3 cartilage-replacement rudiments. Histological changes in the grafts of
talpid3 coracoid and scapula illustrate the crucial stages in the failure of cartilage replacement by bone in talpid* embryos. Initially, the chondroblasts resemble those in the normal rudiment in that they are in the 'young' stage, with
cell expansion just beginning (Plate 1, fig. B). An important difference from the
normal is that the talpid* chondroblasts are not orientated with their long axis at
right angles to the axis of the rudiment. Small areas are found where chondroblasts orientated in that way can be found, but in the greater part of the cartilage
the cells are disorientated and tend to have a round nucleus and to be more
rounded up than in the normal (Plate 1, figs. F, H).
Later, the centre of the talpid3 rudiments (e.g. in the 9-day talpid3 scapula:
Plate 3, fig. R) begins the processes of hypertrophy, with the chondroblasts
becoming positive for glycogen but devoid of phosphatase activity, while in the
remainder of the rudiment chondroblasts are still in the ' young' stage and are
disorientated. Both the disorientation and the hypertrophy are identical with
those found in a scapula removed from a 9-day living talpid3 embryo (Plate, 3,
fig. P). Both grafted and embryonic talpid3 scapulae at this stage have an
irregular shape which contrasts with the regular shape of the normal (plate 3,
fig. O).
In spite of the 'disorientation' in the 'young' stage, talpid3 cartilage often
continues hypertrophy to a stage which is indistinguishable from normal hypertrophic cartilage. This is shown in the 11-day talpid3 scapula (Plate 3, fig. T)
and later in the 14-day scapula where cartilage reaches the last stage of hypertrophy and chondroblasts are positive for both glycogen and alkaline phosphatase. By 14 days in some elements (e.g. the scapula: Plate 4, figs. W, Y) the
EXPLANATION OF PLATES
Abbreviations: B, bone; C, cartilage; Cl, clavicle; Co, coracoid; Er, erosion; Ex, extrusion;
F, fibroblasts; H, hypertrophic cartilage (HI, negative for alkaline phosphatase; HI, positive for alkaline phosphatase); M, undifferentiated mesenchymal cells; Ob, osteoblasts;
Oc, osteocytes; Pc, perichondrium; Po, periosteum; S, scapula.
PLATE 1
Figs. A and C. Adjacent sections of 9-day normal shoulder-girdle graft. Fig. A stained with
haematoxylin and eosin; fig. C stained for alkaline phosphatase.
Figs. B and D. Adjacent sections of 9-day talpid3 shoulder-girdle graft. Fig. B stained with
haematoxylin and eosin; fig. D stained for alkaline phosphatase.
Figs. E and F. Sections of 9-day coracoids from shoulder-girdle grafts, stained with haematoxylin and eosin. Arrow indicates main axis of cartilage rudiment. Fig. E. normal—note
orientation of chondroblasts at right angles to coracoid main axis. Fig. F. talpid3—note
rounding and disorientation of chondroblasts.
Figs. G and H. Fig. G, orientated 9-day normal chondroblasts from coracoid infig.E. Fig. H,
rounded and disorientated 9-day talpid3 chondroblasts from coracoid in fig. B.
/. Embryol. exp. Morph., Vol. 19, Part 3
J. R. HINCHLIFFE&D. A. EDE
PLATE 1
facing p. 332
/. Embryol. exp. Morph., Vol. 19, Part 3
PLATE 2
100//
M
N
Figs. I and J. Sections of 9-day scapula from shoulder-girdle grafts stained for alkaline phosphatase. Fig. I, normal (from graft in Plate 1, fig. C)—inner layer of perichondrium is intensely positive for alkaline phosphatase. Fig. J, talpid3—alkaline phosphatase absent from
perichondrium.
Figs. K-N. Perichondrium of 9-day (K, L) and 11-day (M, N) coracoids from shoulder-girdle
grafts, stained with haematoxylin and eosin. Figs. K, M, normal—K from graft in fig. A
shows beginning of basophilia in cytoplasm of mesenchyme cells adjacent to the cartilage,
and M shows osteoblasts characterized by cytoplasmic basophilia. Figs. L, N, talpid3.
J. R. H1NCHLTFFE&D. A. EDE
/. Embryo!. exp. Morph., Vol. 19. Part 3
PLATE 3
Ift
All slides stained haematoxylin and eosin.
Fig. O. Normal 9-day scapula, from living embryo.
Fig. P. Talpid3 9-day scapula from living embryo.
Fig. Q. Normal 11-day scapula from graft. Note erosion of cartilage and formation of bone.
Fig. R. Talpid3 9-day scapula, from graft.
Fig. S. Normal: bone replacement in the fig. Q scapula. Note osseous material enclosing
osteocytes.
Fig. T. Talpid3 11-day scapula from graft. No cartilage erosion or bone formation.
Fig. U. Talpid3: edge of fig. T scapula. Note 'perichondrium' is fibroblastic.
J. R. H1NCHLIFFE&D. A. EDE
/. Embryol. exp. Morph., Vol. 19, Part 3
• .".Y
J. R. HINCHLIFFE&D. A. EDE
Talpid3 abnormalities
333
3
talpid chondroblasts are fully hypertrophic in an apparently normal way: they
are swollen, possess vacuolated cytoplasm and both they and the matrix are
intensely positive for alkaline phosphatase. However, in some elements, the
cartilage remains in a stage of 'arrested hypertrophy' in which chondroblasts
remain small and do not develop alkaline phosphatase activity. Both types of
cartilage may occur in the same graft, as illustrated in Plate 4, figs. W, Y.
The talpid3 'perichondrium' shows none of the changes undergone by the
normal perichondrium when differentiating into a periosteum. Initially, the
talpid3 'perichondrium' consists of elongated fibroblasts on the outside, and on
the inside of apparently undifferentiated mesenchymal cells with slightly
rounded nuclei and less cytoplasmic basophilia (Plate 2, fig. L) than the osteogenic cells of the normal perichondrium. There is a dramatic histochemical
difference from normal: the talpid3 'perichondrium' shows almost no sign of
alkaline phosphatase activity (Plate 2, fig. J). Later the talpid3 'perichondrium'
remains fibroblastic (Plate 2, fig. N), without any sign of alkaline phosphatase
activity or of the cytoplasmic basophilia characteristic of the osteoblasts found
in the normal at this developmental stage (Plate 2, fig. M). The talpid3 'perichondrium' retains its fibroblastic condition even where it adjoins hypertrophic
cartilage, as is illustrated by the 11-day scapula at the stage when erosion is
occurring and bone is forming in the normal scapula (Plate 3, compare figs. T
and U with Q and S). Later, at 14 days, the 'perichondrium' shows no sign of
mineralization, but it gives a positive response for neutral mucopolysaccharide
though this material is fibrous and not compact as in normal osseous matrix.
DISCUSSION
Autonomy of talpid3 bone formation
Griineberg (1963) has classified systemic disorders of the skeleton into two
types. The mechanism disturbed by the gene in question may be in the skeletal
cells themselves, or it may be of a humoral type affecting these cells wherever
and whenever they occur in the skeleton. In the former case the abnormality is
autonomous, and a transplant will differentiate according to its prospective fate
as determined by its own genetic constitution, while in the latter a transplant
will 'recover' to develop normally.
Talpid3 shoulder-girdle grafts made at the time when the shoulder-girdle
PLATE 4
Figs. V and X. Adjacent sections from 14-day normal shoulder-girdle graft, showing bone
replacement. Fig. V. stained with haematoxylin and eosin, fig. X for alkaline phosphatase.
Fibs. W and Y. Adjacent sections from 14-day talpidz shoulder-girdle graft: fig. W stained
with alcian blue and Mayer's acid haemalum, fig. Y for alkaline phosphatase. The scapula is
fully hypertrophied and is positive for alkaline phosphatase, the coracoid is in 'arrested
hypertrophy' and is negative for alkaline phosphatase. Neither element shows bone-replacement of cartilage.
334
J. R. HINCHLIFFE & D. A. EDE
region is represented by a single pre-cartilaginous mesenchymal condensation,
differentiate according to their genetic fate. There is no recovery of boneforming potential by the coracoid and scapula, even when the grafts are grown to
an age (14 days) which they would not have attained in situ in a talpid3 embryo.
This evidence strongly favours the view that the mechanism disturbed by the
talpid3 gene and responsible for the abnormality is autonomous; it does not
entirely eliminate the possibility that the talpid3 shoulder-girdle-forming condensation is conditioned by a humoral influence before extirpation, because
although there is no visible abnormality in the tissue at the time of explantation,
the abnormality of the whole talpid3 embryo is visible.
There is an interesting parallel with late surviving homozygotes of the
Creeper mutant of the fowl, which resemble talpid3 embryos in that bonereplacement fails in the limb skeleton and to a lesser extent in the vertebral
column and limb girdles, while membrane bone formation is normal (Landauer,
1933). Hamburger (1941) and Rudnick (1945) established the autonomous
nature of the defect by flank grafting Creeper limb buds and the region of the
Creeper limb field and showing that the donor limbs developed characteristic
Creeper phocomelia.
Talpid3 chondrogenesis
Abnormalities in the cartilaginous skeleton of talpid3 embryos have been
traced back to an abnormal system of precartilaginous mesenchymal condensations involving fusions rather than segregation in the limbs (Hinchliffe &
Ede, 1967) and in the head and the trunk (Ede & Kelly, 1964 a, b). In the case
of the shoulder-girdle cartilage elements the earliest visible effect of the gene is
in cartilage differentiation at the orientation phase, when the chondroblasts are
rounded and orientated at random rather than in transverse rows as in the
normal, both in grafts and in situ. Frequently the chondroblasts pass into a state
of 'arrested hypertrophy' but in some cases full cartilage hypertrophy occurs in
a way which is indistinguishable from normal. The abnormality of talpid3
mesenchymal cells thus expresses itself both in the formation of the membranous skeleton and in the pattern of chondrogenesis, which together help to
account for the widespread malformations of the cartilaginous skeleton noted
by Ede & Kelly (19646) at 11 days.
Failure of cartilage-replacement bone in talpid3
In talpid3 embryos and grafts membrane bones are formed normally, indicating that the gene does not prevent all bone formation, but that it interferes
specifically with some mechanism involved in cartilage-replacement bone formation and absent in membrane bone formation.
At the initiation of bone formation in normal cartilage rudiments the inner
layer of the perichondrium differentiates osteoblasts, rich in cytoplasmic basophilia and alkaline phosphatase, which secrete an osseous matrix which later
Talpid 3 abnormalities
335
undergoes mineralization. Then the osteoblasts differentiate into osteocytes embedded in the bone lamellae and finally the cartilage model begins to be eroded
by mesenchymal cells and replaced by bone.
In the talpid3 shoulder girdle grafts the perichondrium of the coracoid and
scapula remains fibroblastic, there is no differentiation of osteoblasts and consequently bone replacement does not take place. No alkaline phosphatase is
produced in the perichondrium, and this enzyme is thought to play an important
role in bone formation (reviewed by Bourne, 1956; Weidmann 1963), though its
precise role is controversial. Fleish & Bisaz (1962 a, b) think that in bone
mineralization the role of alkaline phosphatase is to remove from bone collagen
a pyrophosphate inhibitor which would otherwise poison the crystal growth of
hydroxyapatite precipitated by nucleation. Phosphatase has also been assigned a
role in bone matrix formation. Neumann (cited Bourne, 1956) thinks it is concerned with the production of collagen fibrils. In talpid3, therefore, absence of
alkaline phosphatase from the perichondrium would in itself interfere with
either the formation of the organic matrix of bone, or its mineralization, or
both.
Does talpid3 cartilage fail to induce periostea! bone formation?
Lacroix (1961) put forward the general hypothesis that cartilage induces bone
formation, in support of which he quotes evidence that a block of cartilage
isolated from a rabbit rib-fracture callus 'elicits a lively ossification' when
grafted under the kidney capsule of a rabbit. He makes the suggestion, following
Weiss (1950) that 'the contact of young connective tissue cells with the ground
substance of the inducing cartilage might transform the cells into osteoblasts'.
More specifically, Fell (1956) suggests that chondroblastic hypertrophy is
important in inducing periosteal ossification in embryonic development, and
cites as evidence the study of the role played by cartilage in abnormal bone formation in the Creeper mutant of the fowl. Most Creeper homozygotes die at
4 days, but a small proportion of' escapers' survive longer, manifesting extreme
phocomelia of the wings and legs. Landauer (1932) found a general retardation
of growth in the homozygote ' escapers' at a time when the limb development
was proceeding rapidly. Formation of membrane bone, as in talpid3, was not
disturbed, but in the limbs cartilage differentiation was retarded (Landauer,
1933). The cartilage was histologically abnormal in that there was initially
failure of zone formation, linked with failure to hypertrophy, and finally absence
of rows of cartilage cells in the epiphyseal proliferative zone. There was frequent
partial or complete fusion of tibia and fibula, or radius and ulna, and periosteal
ossification was completely lacking.
Fell & Landauer (1935) attempted to produce a phenocopy of the homozygous Creeper limb phocomelia by retarding the growth of normal chick
limbs in organ culture in a growth-restricting medium. They found that only a
small proportion of limbs grown in the growth-restricting medium showed
336
J. R. HINCHLIFFE & D. A. EDE
ossification and that there was 'invariable association of the ossification with
hypertrophic cartilage'. That the growth-restricting medium did not in itself
directly prevent bone formation was shown by normal ossification of prospective
mandible tissue (a membrane bone) when grown in the growth-restricting
medium. They concluded that' suppression of periosteal bone formation in vivo
(phokomelia) or in vitro is a secondary effect of those agencies (retardation of
growth rate) which prevent cartilage hypertrophy', and advanced the hypothesis
that periosteal bone formation is induced in the perichondrium by the underlying hypertrophic cartilage.
By contrast with talpid3 and Creeper homozygotes, many cases are known of
interference in cartilage organization which do not result in failure to induce
bone. In the fowl, these include sporadic chondrodystrophy (Landauer, 1927),
hereditary chondrodystrophy (Lamoreux, 1942) and the Creeper heterozygote
(Landauer, 1931). In all these cases it appears that the epiphyseal growth cartilage
is histologically abnormal, involving disorganization of the cartilage rows, but
earlier stages of cartilage differentiation appear, or are presumed, to be normal.
In these chondrodystrophies it is reasonable to assume that differentiating
cartilage manages to 'induce' periosteal bone normally, only expressing its
abnormality at a later stage.
More precision can be attached to the phase of hypertrophy held to be responsible for bone induction if more recent histochemical work on the hypertrophy of cartilage is taken into consideration. Fell (1956) associated cartilage
hypertrophy with appearance of alkaline phosphatase in the cartilage, but
Henrichsen's (1958) histochemical work indicates three separate stages in cartilage hypertrophy: first, chondroblast expansion; secondly, glycogen deposition
in the chondroblasts; and finally chondroblast disintegration associated with the
appearance of alkaline phosphatase in the degenerating cells and the cartilage
matrix. In the normal grafted coracoid at 9 days chemodifferentiation of osteoblasts in the perichondrium is already taking place at the time when the chondroblasts of the central zone of the cartilage are in the first stage of hypertrophy, i.e.
cell expansion, but long before the appearance of alkaline phosphatase in the
cartilage. This suggests that, if cartilage induces periosteal bone formation, it
does so during the phase of chondroblast expansion in hypertrophy, and before
the cartilage has reached the stage described on morphological grounds as fully
hypertrophic. This suggestion is compatible with Landauer's finding (1933) that
in the homozygous Creeper embryos, cartilage became abnormal at the stage,
immediately preceding chondroblast expansion, of orientation of chondroblasts
at right angles to the long axis of the rudiments. The abnormality at this stage
may have interfered with the 'inductive' influence of the cartilage on the
perichondrium.
Where there is genetic interference with an inductive interaction only experimental work can determine decisively whether the failure is due to loss of
inductive capacity, or to loss of ability to respond, or to both. The hypothesis
Talpid 3 abnormalities
337
that the talpid3 gene interferes with bone 'induction' by cartilage is none the
less attractive, as it would explain why membrane bone forms normally. However, the abnormality of cartilage differentiation in talpid3 is not, as in Creeper
homozygotes, a simple failure to hypertrophy. Some talpid3 cartilage in shouldergirdle grafts remains in 'arrested hypertrophy'; in other cases hypertrophy is
indistinguishable from normal, but even here there is no bone formation. This
finding makes impossible a satisfactory explanation of the talpid3 bone abnormalities on the older view that 'hypertrophic cartilage.. .is always associated
with periosteal ossification and secretes alkaline phosphatase' (Fell, 1956). If,
however, the view is accepted that periosteal ossification starts at the same time
as chondroblast expansion (but well before alkaline phosphatase appearance),
then, as this is the stage at which all talpid3 shoulder-girdle cartilage is
morphologically disorientated, this disorganization may well indicate an
abnormality in talpid3 cartilage which also expresses itself in failure to 'induce'
osteogenic tissue in the overlying perichondrium.
SUMMARY
1. Chorio-allantoic grafts of the prospective shoulder-girdle region of talpid3
embryos show that the membrane bone (clavicle) develops normally but that the
two cartilage replacement elements (coracoid and scapula) show no sign of the
replacement of cartilage by bone.
2. The cartilage-replacement bone failure is inherent in the talpid3 skeletogenic cells at 4 | days.
3. The orientation of ta/jw^chondroblasts is disorganized at the time when, in
the normal, they are orientated at right angles to the cartilage rudiment long axis.
4. Talpid3 cartilage later differentiates into a stage of ' arrested hypertrophy'
or hypertrophies in a way histologically and histochemically indistinguishable
from normal cartilage.
5. The cartilage-replacement bone failure in the grafts is traced back to a
failure of the talpid3 perichondrium in scapula and coracoid at 9 days to differentiate an inner, alkaline phosphatase-rich layer of osteoblasts.
6. Failure of cartilage-replacement bone formation may be a consequence
of the inability of the abnormal talpid3 cartilage to induce osteogenic cells in the
perichondrium.
RESUME
Anomalies du developpement des os et du cartilage chez le mutant depoule Talpid.3
1. Des greffes chorio-allantoidiennes du territoire presomptif de la ceinture
scapulaire d'embryons talpid3 montrent que les os membraneux (clavicule) se
developpent normalement mais que les elements de deux cartilages (coracoide et
scapula) ne presentent aucun signe de remplacement du cartilage par de l'os.
2. L'absence de remplacement du cartilage par de l'os est une particularite
des cellules formatrices du squelette chez les talpid3 a quatre jours et demi.
338
J. R. HINCHLIFFE & D. A. EDE
3. L'orientation des chondroblastes chez talpid3 est desorganisee au stade
ou, chez le normal, les chondroblastes sont orientes perpendiculairement au
grand axe du rudiment cartilagineux.
4. Le cartilage talpid3 se differencie ulterieurement en un etat d"hypertrophie bloquee' ou hypertrophies qui ne sont pas reconnaissables par rapport
au cartilage normal, ni histologiquement ni histochimiquement.
5. L'absence d'os remplacant le cartilage dans les greffes chez le talpid3 est
due a ce que le perichondre de l'omoplate et du coracoide de 9 jours ne se
difference pas en une couche d'osteoblastes riches en phosphatase alcaline.
6. Le defaut de remplacement du cartilage par de l'os pourrait etre une
consequence de l'inaptitude du cartilage anormal de talpid3 a induire des cellules
osteogenes dans le perichondre.
We wish to thank Mr Eric Maddison and Dr A. H. Sykes for providing facilities in the
Poultry Research Department of Wye College, and the Flock Manager, Mr C. Day, for
maintaining the stock.
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{Manuscript received 13 July 1967, revised 7 November 1967)