J. Embryol. exp. Nlorph. Vol. 30, 1, pp. 119-141, 1973
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Printed in Great Britain
The anatomy and development of
brachypodism in the mouse
By H. GRUNEBERG 1 AND A. J. LEE 1
Department of Animal Genetics, University College London
This paper is dedicated to Professor Walter Landauer on
the occasion of his 77th birthday (15 July 1973)
SUMMARY
The autosomal recessive gene for brachypodism (bp) in the mouse affects the appendicular,
but not the axial skeleton. Manus and pes are more severely affected than the rest of the
limbs; the girdles are normal. In digits 2-5, the basal and middle phalanges of the normal are
replaced by a short and thin element which never ossifies properly; by contrast, the terminal
phalanges are normal or nearly so. Up to the 13-day stage, the limbs of brachypods are externally quite normal, but anomalies of the digital blastemata are detectable in the 12-day
embryo already, i.e. before the onset of chondrification. In the 14-day embryo, the overall
length of metapod + phalanges is still nearly normal; but the basal and middle phalanges of
the normal are represented by a single thin element which is absolutely longer whereas the
metacarpale (metatarsale) is correspondingly reduced in length and calibre. This situation is
subsequently reversed by heterogonic growth as the phalangeal element of brachypods grows
very little. The normality of the terminal phalanges is largely due to the fact that, in the mouse,
much of the terminal phalanx is formed directly from membrane and not by replacement of its
cartilaginous 'model' on which it sits like a thimble. Brachypodism is thus due to an abnormality of the limb blastemata which precedes chondrification, but whose nature is unknown.
Generally, muscular and tendinous anomalies parallel those of the skeleton.
INTRODUCTION
In 1956, the senior author received through the courtesy of the Rockefeller
Institute for Medical Research (as it then was) a breeding nucleus of the gene for
brachypodism in the mouse (bp; linkage group V, chromosome 2; Landauer,
1952) which has been propagated in this laboratory ever since. As the manifestation of brachypodism is regular and homozygotes are viable and fertile in both
sexes, the gene is favourable for developmental studies and it was, for that reason,
included in a series of investigations on the skeleton of the mouse; by the time
this was summarized (Griineberg, 1963), progress on brachypodism had been
slow for a variety of reasons (including a false trail followed too far), and work on
the mutant was interrupted. Meanwhile, an embryological study based on histo1
Authors' address: Department of Animal Genetics, University College London,
Wolfson House, 4 Stephenson Way, London NW1 2HE, U.K.
120
H. GRUNEBERG AND A. J. LEE
chemical methods which was begun in this laboratory has been published by
Milaire (1965), as also some work on a different allele (Ginter & Konyukhov,
1966; Konyukhov & Ginter, 1966; Konyukhov & Bugrilova, 1968; Bugrilova &
Konyukhov, 1971). This somewhat belated publication seems justified as it has
now become clear that brachypodism is due to an embryological modality which
is distinct from the others which have so far been uncovered in inherited mammalian limb anomalies, and as brachypodism has counterparts in man whose
development cannot be studied directly.
Anatomy of the skeleton
Superficially, the skeleton of the bpjbp mouse resembles that of the dachshund
in that a normal skull and axial skeleton is associated with striking limb anomalies. However, in the dachshund it is mainly the long limb bones which are
shortened whereas manus and pes are comparatively normal. In the brachypod
mouse, the situation is reversed and hands and feet are affected more severely
than the rest of the limbs.
Skull and vertebral column are quite normal, nor are there significant
differences in the number of presacral vertebrae (Table 1); though perhaps a
Table 1. Number of presacral vertebrae in brachypods and
normal litter mates (sexes pooled)
26
25±
25
Total
Normal ( + /bp)
11
2
0
13
Brachypod (bp/bp)
8
3
2
13
reduction might emerge in a larger sample. In the forelimb, scapula and clavicula
are normal (Fig. 1), and humerus, radius and ulna are moderately shortened
without any gross abnormalities. The pelvic girdle is normal, except for the
acetabulum, which is shallower and tends to face more posteriorly. The femur is
considerably red uced distally; the condyles are rudimentary and the intercondylar
fossa is shallow or absent. The corresponding condyles of the tibia are similarly
reduced, and the caput tibiae tends to be curved backwards. The fibula often
remains a separate bone not joined to the tibia. It may be of normal length or
Table 2. Length (mm) of normal and brachypod limb bones
130-day-old mice (sexes pooled). Based on data of Landauer (1952)
Humerus
Ulna
Femur
Tibia
Normal (N)
(n = 49)
Brachypod (A)
(n = 50)
A/N
13 03
14-99
16-35
1908
11-92
14-26
13-26
17-19
0-91
0-95
0-81
0-90
121
Brachypodism in the mouse
+ hp
+ hp
hp hp
hp hp
+ihp hpip +;hp hp,hp
5 mm
Fig. 1. The girdles and long limb bones of a normal (+/bp) and a brachypod
(bp/bp) 3, Utter mates, 326 days old. Camera lucida drawings of bones prepared by
the papain maceration technique.
nearly so, but often it is shortened proximally; when this is marked, the caput
tibiae is bent backwards as if the fibula exerted a pull on the tibia. The backward
curvature of the caput tibiae is associated with moderate to severe luxation at
the knee-joint (see below).
Measurements (Table 2) show that both in the fore- and in the hindlimbs the
proximal segment is more affected than the distal one, and that the forelimbs are
less affected than the hindlimbs. However, this semblance of regularity disappears when manus and pes are also taken into consideration:
Forelimb
Scapula, clavicula
Humerus
Ulna
Manus
Hindlimb
Os innominatum
Femur
Tibia
Pes
Evidently, the pattern of involvement of the brachypod limb skeleton cannot be
described in terms of simple gradients. This becomes even more obvious when
manus and pes are examined in greater detail.
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H. GRUNEBERG AND A. J. LEE
Fig. 2. Dorsal view of the right manus of a normal and a brachypod ?, 19 days old.
The arrows point to accessory osseous elements. The left manus of the same brachypod ? is shown in greater detail in Fig. 3. Camera lucida drawings of alizarin clearance
preparations.
The most striking anomalies occur in manus; though there are some variations
between animals, the two sides tend to resemble each other. The hand as a whole
is splayed and the middle digits are bent in an ulnar direction (Fig. 2). Presumably as the result of lack of wear, the claws are long and curved. The proximal
carpals are little affected, but the small distal ones are sometimes difficult to
identify. Metacarpalia 3 and 4 (the longest in the normal hand) are least reduced
in size and least abnormal in shape. Metacarpale 2 (distinctly shorter in the
normal) is usually a misshapen lump though recognizable as a metacarpal by its
position. In digits 1 and 5 metacarpalia can no longer be identified by morphological criteria. Near the base of both of these digits there is an accessory bone
(Landauer, 1952) whose homology is not immediately obvious though some
relationship to the metacarpalia of these digits suggests itself; a study of the
muscular anatomy lends support to this view. Digits 2-5 typically have two
phalanges only. The terminal one is essentially normal in size and shape (see also
Fig. 13), the proximal element is a thin rod of somewhat variable shape which
Brachypodism in the mouse
1 mm
Fig. 3. Dorsal and volar views of the left manus of the same brachypod $ as in Fig. 2.
Fig. 4. Volar views of brachypod digits (manus). (a) 4th right; (b) 2nd right; (c) 3rd
left; (d) 3rd right; (e) 4th left; (/) 3rd left, (b), (d) and (e) 19 days, (a) 101 days, and
(c) and (/) 124 days old.
123
124
H. GRUNEBERG AND A. J. LEE
Fig. 5. Volar views of brachypod digits (manus). (a), (c) and (d)2nd right; (b) 2nd left
(reversed); (e-h) 5thleft. 0 , b, e,h)\9 days, (d) and (g) 101 days, (c) and(f) 124 days
old.
does not resemble either the basal or the middle phalanx of a normal mouse.
Embryological evidence to be discussed below leaves no doubt that this rod is
the equivalent of both these phalanges.
More detail is shown in Figs. 3-5. The basal element of the digits is rod-shaped
distally and tends to be broad and flat proximally where it is also often forked or
bifid. It may be a smooth bone (Fig. 4a-c) or show suggestions (d, e) that it
represents both the basal and middle phalanges of the normal; occasionally two
separate elements are present (/). The two metacarpo-phalangeal sesamoids may
persist as separate bones (c, / ) ; they may be attached by stalks to the basal
phalangeal element (d) or the fusion may be more intimate which leads to
Brachypodism
in the mouse
125
forked and bifid configurations (particularly in the pes); the two sesamoids may
also fuse with each other (Fig. 4a,b,e; Fig. 5 a, b). The pattern of fusions between
sesamoids and basal phalanges shows a curious symmetry with respect to the
middle digit of the pes, which seems to be correlated with a similar pattern in the
abnormally distributed perforated flexor tendons. Indeed, in these as in other
details, brachypods are variable though the overall picture is remarkably
uniform.
In digit 1, in both manus and pes, there is normally a single short proximal
phalanx which, in manus, becomes almost vestigial, as does the first metacarpal.
These reductions are paralleled in brachypods, and indeed there is usually no
positively identifiable proximal phalangeal bone in digit 1 of the manus. The
homology of the accessory bones next to digits 1 and 5 is still problematical.
Fig. 6. Dorsal views of right pes of a 19-day-old brachypod $ and of a normal mouse.
Accessory element next to digit 1 indicated by arrow.
126
H. GRUNEBERG AND A. J. LEE
1 I
2
3
A
\l
I
5
I
Y
- 0r
C.J
Fig. 7. Dorsal views of the right tarsal region of a 19-day-old brachypod 3 and of a
normal 9. Note fusion between centrale (c) (= naviculare) and tarsale 3 (= cuneiforme 3) along with the general reduction in calibre of the metatarsalia. A = talus;
C = calcaneus.
Fig. 8. Volar views of the same feet as in Fig. 7. The accessory element (arrow) is
proximally fused with metatarsale 2. A = talus; C = calcaneus.
Brachypodism in the mouse
127
They may well include material which normally would have contributed to
several elements, particularly to the corresponding metacarpalia and perhaps,
in some cases, to neighbouring sesamoids.
The abnormalities of the brachypod pes (Fig. 6) are similar to those in manus,
but less extreme and less variable in the details from animal to animal. Pes is
less splayed than manus. Metatarsalia 2, 3 and 4 (which are biggest in the normal
foot) are least reduced in brachypod; reduction of digit 5 is less extreme than in
manus, and there is no accessory element next to it. The reduction of digit 1 is
also less extreme, and the accessory element may well be mainly (if not entirely)
the equivalent of metatarsale 1. The end phalanges are normal. In place of the
basal and middle phalanges, there is a single element which tends to be more
regular in shape than the corresponding bones in manus. The metatarsophalangeal sesamoids are often attached to it, but fuse less often with each other.
The tarsalia (Figs. 7, 8) are comparatively normal. Talus, calcaneus and tibiale
are not grossly abnormal though somewhat reduced in size. Various fusions
between the other tarsalia are commonly found. But, as similar fusions are
a feature of many genes affecting the limb skeleton as well as of certain strains
of 'normal' mice, no attempt has been made to study this aspect of brachypodism in more detail.
Muscular anatomy
No attempt has been made to provide a detailed and exhaustive account of the
muscular anomalies; consequently, the number of specimens chosen was the
minimum which appeared to give a clear picture of the major disturbances.
Fore- and hindlimbs of four bp/bp mice, male and female, were used, and of two
heterozygotes. For 'normal' anatomy, (CBA/C57BL) F2 animals were dissected.
Greene's (1935) description of rat musculature is applicable to the mouse, with
the corrections noted by Carter (1951) and Kadam (1962). In general the muscular abnormalities parallel those of the skeleton, the major effects occurring in
the manus and distal antebrachium, in the distal femoral region, crus and pes.
(a) Forelimb
M. triceps brachii caput longum is hypertrophied, its profile at the elbow being
quite different from that in normal mice. There may be additional superficial
fibres on the lateral surface, running across the main fibres, and inserting into
fascia covering the caput laterale.
Mm. extensores carpi radiales (longus et brevis). Their tendons normally insert
on conspicuous tubercles on the radial sides of the second and third metacarpals,
respectively. In brachypod the muscle bellies appear normal, but the tendinous
insertions become much more diffuse, and show a distinct tendency to migrate
laterally. Brevis may even insert fairly strongly on the base of metacarpale 4.
The digital extensors. Corresponding to the distorted form of the brachypod
manus, connective tissue structures on the dorsum of the manus are abnormally
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H. GRUNEBERG AND A. J. LEE
Accessory tendon
to extensor
retinaculum
(b)
(c)
Fig. 9. Diagrams showing the structure of M. extensor digitorum communis in right
forelimbs of normal (a), and brachypod (b-d)mice. Muscle is stippled, tendons solid
black. An additional tendon inserting on the extensor retinaculum is hatched in
(b) and (c).
arranged and lead to disturbances in the terminal distribution of the long tendons.
In the case of the common extensor the anomalies extend proximally to the
muscle itself, affecting its proportional division into individual muscle bellies.
The ulnar extensors, supplying digits 4 and 5, are unaffected.
M. extensor digitorum communis (Fig. 9). In the normal mouse this consists of
four quite separate superficial tendons and separable muscle bellies. In addition a fifth deeper tendon to digit 3 arises from the ulnar margin of the muscle.
In brachypod there are usually fewer main tendons, due to the absence or
reduction of a tendon to digit 3 or 4, but smaller accessory tendons are frequently present, which either fuse distally with a main tendon, or become lost
in loose connective tissue. The most radial tendon bifurcates to supply digit 1
as well as 2, and may give off a thinner tendon to digit 3. A further anomalous
tendon, usually but not invariably present, arises from the most radial belly and
inserts into the extensor retinaculum.
M. extensor digitorum profundus (vel indicis proprius). The muscle belly is
normal or slightly reduced. Its tendon diminishes rapidly as it approaches the
Brachypodism in the mouse
129
Fig. 10. Diagrams of the tendons of M. flexor digitorum superflcialis (black), and
M. flexor digitorum profundus (unshaded) in the right manus of normal (a), and
brachypod (b, c) mice.
carpus, where it becomes lost in a fan of loose connective tissue spreading over
the bases of all metacarpals, but concentrated on numbers 3 and 4.
The digital flexors. Except for the radial part of M. flexor digitorum superficialis, the muscle bellies are unaffected. The perforating tendons, inserting on
the virtually unaffected terminal phalanges, are essentially normal, except that
to digit 2. However, the perforated tendons, normally inserting on the second
phalanges of the middle three digits, are highly abnormal in both form and
attachments. On the radial side, the region overlying the point of origin of the
first and second perforating tendons forms a focus for multiple anomalies involving subdivision, suppression and fusion of deep and superficial tendons (Fig. 10),
and affecting the first lumbrical muscle.
M. flexor digitorum superficialis. The radial tendon, in addition to supplying
digit 2, usually gives off a strong branch which joins the deep tendon to digit 1,
and another which attaches to the flexor tendon sheath of the latter. The
additional tendon to digit 1 may arise from a separate muscle belly, closely
associated with that supplying digit 2. The distal ends of the perforated tendons
are weakly formed where they loop round the deep tendons, and after reuniting
they insert on the metacarpophalangeal sesamoids and basal phalangeal elements.
M. flexor digitorum profundus. The tendon to digit 2 is usually misshapen and
may be absent at its origin, in which case the distal perforating tendon is a
continuation of one or more of the multiple tendons from the radial part of
M. flexor digitorum superficialis. Otherwise, apart from fusions with the superficial flexor tendons already mentioned, this muscle shows no abnormalities.
M. flexor carpi radialis. The muscle belly is normal but its tendon gains
insertion into the bases of the 2nd to 5th metacarpals through a thick ligamentous
arc.
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E M B 30
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H. GRUNEBERG AND A. J. LEE
Mm. lumbricales. The 2nd to 4th lumbricals are well developed but their
tendons are greatly expanded in a vertical plane, inserting widely into connective
tissue over the medial surface of the distal ends of their respective digits. The
4th lumbrical extends immediately radial to the supernumerary metacarpal of
the 5th digit. The 1st lumbrical is reduced in size and in one specimen inserted
on the distal end of the 1st digit; it may be subdivided, one part originating from
the radial tendon of M. flexor digitorum superficialis. In one specimen a few
fibres, evidently derived from the 4th lumbrical, inserted into the perforated
tendon to digit 4, a situation resembling the Mm. interflexorii of the normal pes
(see below).
In the manus itself connective tissue investing the carpus, metacarpus, and
digits is much denser and more diffuse, and normally discrete ligaments become
difficult to recognize. The short intrinsic muscles, however, are surprisingly little
affected. Apart from the absence of an adductor to the 1st digit (much reduced
even in normal mice) and an incomplete separation of the remaining two adductors from the underlying Mm.flexores brevesprofundi (interossei), all remaining
muscles are identifiable, although understandably much reduced.
(b) Hindlimb
The abnormal acetabulum has already been mentioned; this may be a secondary post-natal effect associated with abnormal gait and posture. At all events,
resultant changes in the orientation of the head of the femur are apparently the
reason for the development of numerous relatively massive sesamoids in the
tendons of the Mm. gemelli and obturator internus.
The shortening of the femur leads to only minor changes in the insertions of
the adductor group of muscles. The reduction in the area available for muscle
attachments on brachypod femora is accompanied by compensatory marginal
expansions of thick connective tissue ridges, anchored distally to the medial and
lateral fabellae.
The poor development of the femoral and tibial condyles is associated with a
variable luxation at the femoro-tibial joint. When this is severe, the end of the
femur becomes displaced distally behind the caput tibiae, articulating with the
posterior surface of the latter, and even pressing against the head of the fibula.
The joint cavity extends correspondingly behind the tibia, with hypertrophied
irregular menisci containing many sesamoids. Its connective tissue capsule is
massively thickened and prevents the normal attachment of neighbouring
muscles. The tendon of origin of M. soleus is partly confluent with the abnormally extended joint capsule, and partly with thickened fascia overlying M.
popliteus and adjacent muscles, and may contain a large sesamoid. M. popliteus
is inevitably affected by the dislocation of the knee joint, its tendon frequently
becoming vestigial or lost in connective tissue, and its muscle fibres are often
partially disarranged, presumably through loss of its normal function. Other
muscles with abnormal attachments in the region of the knee include M.
Brachypodism
in the mouse
131
gastrocnemius, the lateral tendon of origin of which has extensive connexions
with the patellar ligament, and M. extensor digitorum longus, which has an
accessory fleshy head originating on the caput tibiae.
In the crus, muscular abnormalities are no more than one would expect from
the shortening of tibia, and especially, of fibula.
Anomalies in the pes are more complex, but although showing slight similarities to those in the manus, are much less severe than the latter. As in the forelimb,
the terminal distribution of the digital extensor tendons is disturbed, particularly
those to digits 2 and 3. The two separate Mm.peronei to digits 4 and 5 are present
in brachypods as in normals, but a tendon from M. extensor digitorum longus
to digit 4 tends to be lacking. Characteristic of brachypod is an extra deeper
tendon from the long extensor, running between the 3rd and 4th metatarsals and
terminating mainly on digit 3, but branching also to digit 4. That this is a
different entity from the missing tendon to digit 4 is indicated by the fact that it is
not uncommonly present in heterozygotes, simultaneously with a normal set of
tendons supplying digits 2-5. M. extensor digitorum brevis normally consists of
two separate muscles to digits 2 and 3; in brachypod the lateral one is absent,
while the medial tendon tends to fuse with the long extensor. The lateral head is
frequently absent in heterozygotes also, but is sometimes merely reduced, in
which case it joins the extra tendon from the long extensor, a fact which
suggests that this additional tendon in homozygotes may represent material
which would otherwise have contributed to the lateral tendon of the short
extensor.
The insertions of the perforated tendons are similar to those in the manus. In
the pes, however, the terminal bifurcations in digits 2 and 4 tend to be absent
on the side adjacent to the middle digit. This tendency appears to be correlated
with the pattern of fusions between metatarso-phalangeal sesamoids and basal
phalangeal elements: where a tendon slip is retained there is less fusion, or none
at all. Evidently lack of fusion is associated with the possibility of some movement, since, as already mentioned for the manus, the perforated tendons insert
mainly on the sesamoids at the base of the phalanges.
Intrinsic musculature of the pes is minimally affected. The lumbricals insert
into the sides of the distal ends of the digits. Mm. interflexorii in normal mice are
short muscles supplying digits 2-5, originating on the deep flexor tendons and
inserting into the perforated tendons. In brachypod they are restricted to digits 3
and 4, with occasional weak contributions to digit 5.
The development of the brachypod limb skeleton
As brachypod mice are fertile in both sexes, litters from bpjbp x bpjbp matings
are easily obtained. Hence bpjbp embryos can be compared with normals at
stages at which they cannot be distinguished by external inspection. As in
previous studies, normal embryos were obtained from a cross between two
inbred strains ((CBA/Gr x C57BL/Gr) F2). Mice of that genotype are very
9-2
132
H. GRUNEBERG AND A. J. LEE
(a)
(a)
(b)
13 days
12 days
l m m
13-5 days
14 days
Fig. 11. Limbs of normal (solid lines) and brachypod (broken lines) embryos. Tracings
from photographs. In each case (a) is the right fore- and (b) the right hindlimb.
vigorous and have much more uniform and 'normal' skeletons than the parent
strains and indeed most inbred strains of mice.
Up to and including the 13-day stage (Fig. 11), brachypod limbs do not differ
in shape or size from normals as seen in dorsal view or as studied in series of
transverse sections. Brachypod limbs begin to lag behind the normal ones in the
13-5-day stage, and in 14-day embryos, a comparatively modest but clear difference has been established. In the 13-day mouse embryo, the skeleton of manus
and pes consists of well-developed blastemata; in the forelimb, the first signs of
chondrification can be detected in digital rays 2, 3 and 4; none is present yet in
the hindlimbs. In the 14-day stage, chondrification is well advanced in the forelimbs and a little less so in the hindlimbs.
At first glance, the blastemata of the limbs of 12- and 13-day brachypod
embryos differ little from those of normal embryos in their gross features, a fact
also commented on by Milaire (1965). As soon as cartilage has been laid down
in the 14-day stage, striking differences between normal and brachypod are
obvious. We shall here deal with the anomalies of the cartilaginous skeleton
first and return to earlier stages later on.
In manus and pes, metacarpalia and metatarsalia are represented by cartilaginous rods which are shorter than normal and much reduced in calibre
(Fig. 12). Their cartilage histology is essentially normal but clearly retarded.
The phalangeal region is even more abnormal. Instead of the firm basal and
Brachypodism in the mouse
133
Fig. 12. The third digital rays of the left forelimbs of a normal and a brachypod
embryo, 14 days old. met. 3, metarcarpale 3; b.ph., basal phalanx; m.ph., middle
phalanx ;ph.e., phalangeal element corresponding to both basal and middle phalanx
in the normal. Bouin. H. & E. 7-5 /*m. x 100.
middle phalanges in the normal, there is a single pencil-thin element in the
brachypod. At this stage, the overall length of the digital skeleton of the brachypod does not yet differ much from that of the normal. However, whereas in the
normal the metacarpale is longer than the two phalanges, the situation is reversed
in the brachypod where the phalangeal element is both relatively and absolutely
longer than the corresponding metacarpale or the two phalanges of the normal
embryo. It can thus scarcely be doubted that the phalangeal element in brachypod is the equivalent of both basal and middle phalanx in the normal. Presumably in some way the reduced calibre is the reason why it remains a single
element. Attention should here be drawn to the roundish condensation of mesenchyme at the level of the future metacarpo-phalangeal articulation of the
brachypod. The general situation is essentially the same throughout, both in the
14- and in the 15-day embryo.
The relative proportions of the skeletal elements at the time of chondrification
are totally different from those in adult life, when metacarpalia and metatarsalia
134
H. GRUNEBERG AND A. J. LEE
Fig. 13. Sagittal sections through middle digits (hindlimb) of a + \bp (normal) and a
bp/bp (brachypod) mouse; litter mates, 1 day old. met., metatarsale; ses., metatarsophalangeal sesamoid; b.ph., basal phalanx; m.ph., middle phalanx; t.ph., terminal
phalanx; ph.e., phalangeal element corresponding to both basal and middle phalanx
in the normal. Susa. H. & E. x 60.
are much longer than the phalangeal element (Figs. 2 and 6). Though the metapod is short to start with, it is capable of considerable longitudinal growth; by
contrast, the phalangeal element has an initial advantage over the two phalanges
in the normal, but elongates comparatively little thereafter. Heterogonic growth
thus completely reverses the situation, and most of this change happens during
the 3rd week of gestation (Fig. 13).
The histological differentiation of the more abnormal cartilages of brachypods
is retarded and delays ossification for a long time (Konyukhov & Ginter, 1966).
Brachypodism in the mouse
135
At birth (Landauer, 1952), alizarin preparations reveal no sign of ossification in
manus or pes, with the exception of the terminal phalanges which, somewhat
paradoxically, are essentially normal. Eventually, ossification catches up and in
the adult it is complete or nearly so almost everywhere, except in the phalangeal
elements whose immature (eosinophil) cartilage is never completely replaced by
bone (see also Fig. 13).
The normality of the terminal phalanges is not as odd as it appears at first
sight. The original blastema present in the 13-day embryo includes the material
up to and including the middle phalanx as is clearly visible in the 14-day stage
when chondrification has occurred. The terminal phalanges do not arise until a
day later, and this may be the reason why the cartilaginous terminal phalanges
are much better developed than the proximal phalangeal element (Fig. 13).
More important is the fact that in the mouse, the osseous terminal phalanx
is formed not so much by endochondral replacement, but mainly directly from
membrane on top of the cartilage like a thimble; it is thus virtually independent
of its cartilaginous 'model', and not directly derived from the limb skeleton
blastemata.
As mentioned above, as a first approximation the blastemata of the 13-day
brachypod manus and pes appear to be normal. In situations such as this, one is
faced with a technical difficulty. Valid comparisons can only be made between
accurately corresponding sections; e.g. between sections in which the foot plate
is sectioned axially through all the digits and with the marginal blood sinus
cut all round the periphery. This ideal situation is, of course, rarely realized,
and in practice one has to be content with approximations. But, in successive
comparisons between normals and abnormals only fairly striking differences are
likely to be detected. For simultaneous comparisons, the following procedure
has been adopted. All the four limbs of five pairs of accurately matched normals
and brachypods were processed and stained simultaneously; the best-fitting
section in each case was photographed, all the exposures and the printing being
done simultaneously for the whole group. Mounting the photos of the four
limbs of normal and brachypod on one piece of cardboard, simultaneous comparisons become possible, and indeed several pairs can be compared in one
operation. Due to faulty orientation in sectioning two forelimbs and one hindlimb, from three brachypod embryos, are not available for comparison. Of the
five pairs studied, four represent the 13-day stage proper and form a homogeneous group; the fifth pair is slightly more advanced. The validity of the comparisons ultimately depends on the degree of similarity between the right and
left limbs of the same embryo, and that between embryos of the same genotype.
On that background, the reality of differences between genotypes must be judged,
and we have no doubt that those to be described presently are real and not due
to limitations of method and observer. But unfortunately, not all the photos can
be reproduced.
Confining ourselves at first to the forelimb which is more advanced in develop-
136
H. GRUNEBERG AND A. J. LEE
Fig. 14. The right limbs of a normal (above) and a brachypod embryo (below),
13 days old. Forelimbs on the left, hindlimbs on the right. Bouin. H. & E. 7-5 /mi.
x35.
ment (Fig. 14), four differences in the blastemata of the middle digits have been
detected. (1) The brachypod blastemata are slimmer, particularly distally where
in the normal embryo some flaring of the blastema next to the marginal blood
sinus is generally visible; this is much less marked in brachypods. (2) In the
normal, a distinct bulge of the digital blastemata is present about halfway
between carpus and limb margin; it indicates the position of the future
metacarpo-phalangeal joint. In the brachypod, the blastemal outline is more
Brachypodism
in the mouse
137
spindle-shaped, with its greatest width more proximal than in the normal. (3) In
the brachypod, a condensation of mesenchyme corresponding to the level of the
metacarpo-phalangeal joint later on (Fig. 12) is present; it lies in a slightly
lighter halo of mesenchyme; in the normal, this is not detectable at this stage.
(4) In the brachypod, the blastema of digit 2 is interrupted basally; this is strikingly so in seven hands and slightly in the remaining one available for study.
Scrutiny of serial sections reveals that it is due to curvature of this blastema
which thus cannot be cut axially along its length in a single section. The gross
Fig. 15. The left limbs of a normal (above) and a brachypod embryo (below), 12 days
old. Forelimbs on the left, hindlimbs on the right. Bouin. H. & E. 7-5 /tm. x 35.
138
H. GRUNEBERG AND A. J. LEE
abnormality of metacarpale 2 in the adult (Figs. 2, 3) will be remembered. In
summary, all the anomalies of the brachypod manus are already detectable in
the 13-day stage when chondriflcation is only just beginning. In the hindlimbs
(Fig. 14), skeletal development is less advanced; the digital blastemata are still
approximately cylindrical and there is no sign of chondrification yet. However,
the brachypod blastemata are consistently slimmer and the intervening intervals
correspondingly wider than in the normal foot plate.
The material available for the 12-day stage is less complete (normal, CRL
6-9 mm; bpjbp, CRL 7-4 mm; all four limbs each; and normal, CRL 6-1 mm;
bpjbp, CRL 7-5 mm; left fore- and hindlimbs only). However, the situation is
consistent within the group and in agreement with the findings in 13-day
embryos (Fig. 15).
The digital blastemata of the brachypods are consistently slimmer and the
intervening intervals between them correspondingly wider than in the normal
embryos. It must be concluded that the limb anomalies of brachypods are
present in the blastemata prior to chondrification. The brachypod syndrome thus
has nothing to do with chondrification as such which merely makes an existing
anomaly strikingly visible. This conclusion is not surprising, as brachypod
embryos produce completely normal cartilage elsewhere and must therefore be
histogenetically competent so far as that tissue is concerned.
No attempt has been made to examine the 11-day stage as in the limb buds
of that stage no blastemata are detectable yet.
DISCUSSION
Among the skeletal genes of the mouse (Griineberg, 1963), some act systemically and affect the skeleton as a whole. Other genes have localized effects: they
interfere with processes which are confined to certain localities of the body,
but do not come into operation elsewhere. Thus genes which single out the
axial skeleton have been shown to affect either the notochord (T, Sd, Pt, tc)
or the paraxial mesoderm and particularly the formation and differentiation of
the sclerotomes (vt, Bn, Rf, pu, Lp, Cd, Fu, tk, un and others). Genes which
single out the appendicular skeleton involve processes peculiar to the limb buds.
As a group, these are less well understood. Some of these genes affect size and/or
shape of the limb buds in early stages before blastemata have been formed;
subsequently, when these arise, the mesenchyme seems to be used according to
the same rules as in normal development, so that an excess may lead to polydactylism, a deficiency to oligodactylism, and a narrowing of the foot plate to
various types of syndactylism. In all these cases it appears that the digital
blastemata formed are essentially normal at first though a variety of complications usually supervenes later on. By contrast, in brachypod size and shape of
the foot plates is at first quite normal, and the first anomalies which have been
discovered involve the blastemata which are demonstrably reduced from the
Brachypodism in the mouse
139
beginning. It is improbable that the gene for brachypodism acts simply by reducing the size of the blastemata. Almost any disturbance of a developmental
process tends to retard the growth of the structure affected; reduction in size is
thus probably an unspecific concomitant of a variety of different derangements;
but it tends to be the earliest disturbance detectable by morphological methods.
In the present case, as generally in such situations, the ultimate nature of the
disturbance remains to be discovered.
However, given the blastemal anomaly, the ensuing syndrome can be understood fairly clearly. In manus and pes, the material destined to form metapod
and phalanges is subdivided anomalously so that too little is allocated to the
metapod and too much to the phalanges. Apparently a similar though less
extreme process has happened at an earlier stage of limb development. In both
limbs, the stylopod is more strongly reduced than the zeugopod, so that the
latter becomes relatively longer than normal. If this reflects mis-allocation of
material rather than differential growth (which has not been directly observed),
the stylopod (like the metapod) would have received too little and the zeugopod
(like the phalanges) too much of the original blastema. If so, the only difference
would be that whereas in manus and pes the original situation has been reversed
by differential growth, this has not happened in the proximal region. The
bshaviour of the terminal phalanges has already been discussed.
The remainder of the skeletal anomalies in brachypods involves mainly
anomalous fusions between cartilages and bones. These are probably secondary
events of little significance. In normal development, adjacent cartilages commonly undergo temporary fusions which are subsequently resolved again, such
as atlas-axis fusion (Dawes, 1930) or fusions between carpalia and tarsalia
(Whillis, 1940). Such processes are easily disturbed with the result that, e.g.
permanent atlas-axis fusions are common in undulated (unjuri) and not rare in
C57BL/Gr mice; and that fusions between carpalia and tarsalia accompany
many genetic anomalies of the feet and are also found as isolated entities in
certain inbred strains of mice (for details and references see Griineberg, 1963).
It has also been shown that local pressure easily leads to anomalous fusions
between cartilages (Gliicksmann, 1938, 1942). A more detailed study of these
phenomena in brachypod mice would thus scarcely be profitable.
It is obvious that some, at least, of the tendinous anomalies are secondary to
the skeletal deformities of brachypodism. For instance, the perforated tendons,
in the normal mouse, insert on the second (middle) phalanx of the digits
concerned, but in bpjbp where there is no separate middle phalanx, they insert
more proximally. Whether all tendinous anomalies may be regarded as secondary is an open question, as is also the relationship between muscular and
tendinous abnormalities. This doubt arises mainly from the tendinous anomaly
observed in three out of four hindlimbs of + \bp heterozygotes in the (presumed)
absence of osseous anomalies. Some features like the hypertrophy of the M.
triceps may be the result of posture (bpjbp mice with their reduced limbs using
140
H. GRUNEBERG AND A. J. LEE
that muscle to raise their bodies off the ground); if so, this would be a somewhat
remote gene effect.
In his histochemical study of the development of brachypodism, Milaire
(1965) came to the conclusion that bp affects early chondrogenesis without in the
least involving morphogenesis. ('Le brachypodisme est une mutation qui affecte
les evenements initiaux de la chondrogenese primordiale. L'alteration porte a la
fois sur la condensation primaire des cellules precartilagineuses, sur leur aptitude
a synthetiser la substance fondamentale mucoproteique, sur la croissance des
jeunes cartilages et sur la chronologie de leur ossification.') This concept does not
account for the localization of the anomalies in the limbs, as chondrogenesis
elsewhere is quite normal. The discovery that the blastemata are reduced prior to
chondrification puts the blame on a skeletogenic mechanism peculiar to the
limb skeleton whose chondrification is thus only secondarily affected. However,
perhaps Milaire knew more than he said; for, although the early blastemal
reductions are not described, he refers in his summary to the condensation of the
precartilage cells though apparently not as the root cause of all the anomalies of
the brachypod limb skeleton.
The mis-allocation of blastemal material as between metapod and phalanges
described in this paper is somewhat similar to the incorporation of material
destined to form the posterior end of the vertebrae into the following intervertebral discs in the undulated mouse (Griineberg, 1954).
Brachypodism is a 'congenital defect'. Are such stationary situations 'burntout' processes which have happened once and for all in early development, or
are there continuing gene effects of an unknown nature which can be detected as
active processes later in life (Gruneberg, Gray & Truslove, 1965)? Brachypodism
was included in a search for continuing gene effects involving 13 mutant genes;
this was based essentially on comparisons of mutant with normal weights
throughout life. At birth, bpjbp mice weigh only a little less (0-956) than their
normal litter mates (Landauer, 1952). During the first month (Gruneberg et al.
1965), they fall back to about 0-72 at 28 days, but eventually stabilize at a higher
level (0-84 in ^ ; 0-88 in $$) for the rest of their lives. Whether, in this particular
case, the deterioration of the brachypods after birth is a direct consequence of
their limb anomalies, or whether it indicates a continuing gene effect, is a
moot point.
The human counterparts of bp are known under the name of brachydactylism
in the wider sense of the word (Bell, 1951; Grebe, 1964); the group includes
many phenotypically and genetically distinct entities; in some of them, the
digital anomalies are accompanied by short stature which indicates that, as in
bp, the limb is affected as a whole. However, closer comparisons are beset with
difficulties. For obvious reasons, only the final state of affairs is known in the
human conditions; moreover, in mammals as distinct as mouse and man, even
strictly homologous genes will presumably often give rise to phenotypes which
differ to a greater or lesser extent. For instance, whereas absence of phalanges
Brachypodism in the mouse
141
(hypophalangism) is found in several human brachydactylisms, the basal phalangeal element of bp stands out by its thinness. At present there is no way of
discovering whether this is so because bp is a gene different from all the known
human ones, or whether bp is homologous to one of them, but acts differently on
a different genetic background. As more murine genes affecting the feet become
available, comparisons will become more meaningful. In the meantime, the value
of the animal material lies in the fact that the origin of anomalies can be followed
step by step by direct observation instead of being a matter of conjecture.
Our thanks are due for technical assistance to Mrs Beryl Mullins (nee Fannon) and for
secretarial assistance to Mrs Marilyn Mendoza.
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(Received 11 January 1973)