/. Embryol. exp. Morph. Vol. 33, 4, pp. 1013-1022, 1975
Printed in Great Britain
1013
Skull growth in achondroplasic (en) mice;
a craniometric study
By ROSEMARY J. JOLLY 1 AND W. J. MOORE 1
From the Department of Anatomy, University of Leeds
SUMMARY
Skull morphology in achondroplasic (cn/cn) mice was compared with that of normal
siblings in order to determine the effects of this chondrodystrophy on skull growth, particular
attention being given to dimensions reflecting growth at the synchondroses of the cranial
base, the nasal septal cartilage and the condylar cartilage of the mandible. The central section
of the cranial base (basicranial axis) was reduced by 25 %, the length of the viscerocranium
by 18 % and the length of the condylar process by 11 %. The evidence indicates that these
reductions are due to diminished growth at respectively the spheno-occipital and midsphenoidal synchondroses, the nasal septal cartilage and the condylar cartilage. The relative sizes of
the reductions in cranial base, viscerocranium and condylar process suggest that the growth of
synchondrotic and septal cartilages is diminished to a greater extent than that of condylar
cartilage. This finding is in agreement with the observations that condylar cartilage, unlike
synchondrotic and septal cartilage, grows by surface apposition and that the principal
defect in enjen mice is a disturbance of interstitial cartilaginous growth. The posterior
extension of the basicranial axis of the cn\cn mice was reduced by 14 % and the anterior extension by 2 %. The width of the cranial base was decreased by 9 % and the angle between
the basicranial axis and its anterior extension was decreased by 3 %. The length of the
neurocranium was reduced by 19 % in the cn\cn animals while the volume of the endocranial
cavity was diminished by only 18 %. The latter reduction is less than would be expected from
the cube relationship between volume and linear dimensions but is readily accounted for by
the lack of reduction in the height or width of the neurocranium, the slight flattening of the
cranial base and the doming of the neurocranial vault.
INTRODUCTION
Inherited chondrodystrophies have been reported in a wide variety of vertebrate groups, including chickens, mice, rats, rabbits, dogs, sheep, cattle and
man (for comparative descriptions and bibliography see Griineberg, 1963).
All are associated with disturbances of endochondral ossification that lead to
disproportionate dwarfism in which the limbs are usually shortened to a greater
extent than the trunk. The changes produced in the skull are generally less well
documented than those occurring postcranially, but it appears that in the
majority of the mammalian conditions the cranial base is shortened with compensatory broadening of the calvarium and retraction of the nasal skeleton
(Crew, 1923; Stockard, 1941; Landauer and Chang, 1949; Gruneberg, 1963).
1
Authors' address: Department of Anatomy, School of Medicine, University of Leeds,
Leeds LS2 9NL, U.K.
63-2
1014
ROSEMARY J. JOLLY AND W. J. MOORE
The complex changes in the skull are of particular interest in that this part of
the skeleton contains, in addition to the endochondral elements of the cranial
base and sense capsules, the intramembranously ossifying dermal bones of the
calvarium and face. Moreover, certain of the dermal bones contain zones of
secondary cartilage which appear after the initiation of intramembranous ossification, but subsequently undergo endochondral ossification to make a significant contribution to overall bony growth. The number of secondary cartilages
varies from one vertebrate group to another but includes the condylar, coronoid
and angular cartilages of the mandible in most mammalian groups (De Beer,
1971). Of these, the condylar cartilage has been the most intensively studied in
many species, including the mouse, and its growth has been found to proceed
by apposition on its upper surface as a result of the activity of chondroblasts
differentiating in the intermediate cell zone (located between condylar and
articular cartilages) rather than by interstitial proliferation of chondrocytes such
as occurs in a typical epiphyseal cartilage (Blackwood, 1966; Frommer, Monroe,
Morehead & Belt, 1968; Silbermann & Frommer, 1972). Unlike epiphyseal
cartilage, no regular columns of hypertrophic chondrocytes are formed in the
condylar cartilage (Durkin, Irving & Heeley, 1969) and the cells survive their
passage through the cartilage to emerge, still vital, in the zone of ossification
on its undersurface (Silbermann & Frommer, 1972). In view of the observations
that the underlying defect in many of the mammalian forms of chondrodystrophy
lies in the processes of interstitial cartilaginous growth that lead to the formation
of the columns of hypertrophic chondrocytes (Griineberg, 1963), it might be
expected that these disorders would be associated with less disturbance of the
appositionally growing condylar cartilage than of the interstitially growing
septal cartilage of the nasal capsule or synchondrotic cartilages of the cranial
base.
In this study, a craniometric analysis is made of the effects of one type of
chondrodystrophy (cn/cri) occurring in the mouse upon the growth of the skull
components, with special attention being given to possible differential effects
at the various sites of endochondral ossification. This particular chondrodystrophy was chosen because it has been shown (Konyukhov & Paschin, 1970)
that the cartilage defect consists of a reduction in the rate of chondrocyte
division manifested by a diminution in the number of chondrocytes per isogenous group in zone II (the zone of cellular proliferation) of the epiphyseal
cartilage. It might be expected, therefore, to provide a suitable model for the
skull changes observed in other chondrodystrophies, including the classic form
of human achondroplasia, in which there is a failure of interstitial cartilage
growth.
Skull growth in mice
1015
MATERIALS AND METHODS
Animals. The achondroplasic strain of mice arose as a spontaneous mutation
in the AKR/J strain (Lane & Dickie, 1968). The body weight of cn/cn mice is
reduced at birth compared with normal siblings (i.e. + / + or en/ + ) and growth
retardation continues for the first month of life. From the fifth week onwards,
the relative rates of growth of the cn/cn mice and their normal siblings are
similar (Konyukhov & Paschin, 1967).
The cn/cn mice can be distinguished at birth by their dome-shaped skulls and
short thick tails. Many of the affected animals die, but those that reach maturity
have shortened trunks and tails, pronouncedly shortened limbs, and heads that
retain their initial doming (Lane & Dickie, 1968). A malocclusion, in which the
lower molars occlude anterior to the upper molars, present as soon as
the teeth have erupted, becomes progressively more accentuated as growth
proceeds.
As in most other forms of heritable chondrodystrophy, the fundamental
defect of cartilage growth is unknown, but the work of Konyukhov & Paschin
(1967), using subcutaneous implantation of limb bones between 7- or 14-day-old
cn/cn mice and their normal litter-mates, suggests that there is a primary gene
effect manifested in the chondrocytes and also a growth-inhibiting substance
released into the blood in the cn/cn mice. In a later study (Konyukhov &
Paschin, 1970) these authors found that the principal difference between the
epiphyseal cartilages of the cn/cn and normal mice was a reduction in the
thickness of the hypertrophic zone (zone III) and a reduction in the number of
cells per isogenous group in the columnar zone (zone II). These differences
became less pronounced after 21 days of postnatal life.
Post mortem. Ten cn/cn mice (9 female, 1 male), all more than 13 weeks old,
were compared with 10 similarly aged normal siblings (all female). The animals
were killed by ether inhalation and the skeletons macerated by immersion in
papain solution at 56 °C. Lateral radiographs were taken of the crania, each
being positioned so that the X-ray beam was directed at right angles to the
mid-sagittal plane along the long axes of the external auditory meatuses, with
constant tube-object-film distances.
Biometry. The following measurements were taken on the dried skulls using
a measuring microscope (Fig. 1).
(1) Overall length of cranium, opisthion (a) to most anterior point on nasal
bones (b).
(2) Length of neurocranium, opisthion (a) to nasion (/).
(3) Width of neurocranium, between posterior roots of zygomatic arches
(c-C).
(4) Height of neurocranium, mid-point between fronto-parietal and parietointerparietal sutures (d) to spheno-occipital synchondrosis (e).
(5) Width of cranial base, between lateral borders of carotid canals (g-g').
1016
ROSEMARY J. JOLLY AND W. J. MOORE
FIGURE 1
From above down: ventral and dorsal views of cranium, medial view of mandible of
mouse to show measurements made on skulls. See text for definitions.
(6) Length of viscerocranium, nasion (/) to most anterior point on nasal
spine (b).
(7) Width of viscerocranium, between lateral surfaces of upper second molar
teeth Qi-h').
(8) Height of viscerocranium, nasion (/) to midline of palate at a transverse
axis through the anterior borders of the first molar teeth (J).
(9) Length of mandible (left), condyle (k) to symphysis (/).
(10) Length of condylar process (left), condyle (k) to masseteric notch (m).
(11) Volume of endocranial cavity; the external surface of the cranium was
coated with wax to render it watertight and the volume of water required to fill
the endocranial cavity was then determined using a tuberculin syringe.
Skull growth in mice
1017
FIGURE 2
Diagram of radiographic outline of cranium of mouse to show linear and
angular measurements of cranial base. See text for definitions.
As a measure of the changes in the limb bones, the length of the humerus was
taken between the intertubercular sulcus and intercondylar notch.
Both linear and angular measurements were taken on enlargements of the
radiographs. Since the aim of the study was to compare the normal and
achondroplasic mice, rather than to obtain absolute measurements, no correction was made for magnification, which was identical for both groups. The
linear measurements were related to the three components of the cranial base
and included the following (Fig. 2).
(1) The basicranial axis, presphenoid-ethmoidal synchondrosis («) to the
Bolton point (/?).
(2) The anterior extension, presphenoid-ethmoidal synchondrosis («) to
the fronto-ethmoidal suture (q).
(3) The posterior extension, Bolton point (p) to opisthion (a).
The angular measurement was taken between the basicranial axis and its
anterior extension, as determined by drawing lines tangential to the outlines of
the superior surfaces of the ethmoid bone and of the basicranial axis and
measuring the superior angle at the point of intersection (Fig. 2). To make it
comparable with the spheno-ethmoidal angle as used in man, the angle was
subtracted from 360°.
Means and standard errors were computed for each dimension in the cnjcn
and normal mice. The means for the two groups were compared by t tests,
P values below 0-05 being taken as statistically significant.
RESULTS
Cranial base. The total length of the cranial base was reduced in the cnjcn mice
compared to the normal animals but the amount of shortening was not uniform
in the three subdivisions. The greatest reduction was in the basicranial axis
which was shortened by almost 25 %, while the posterior extension was
shortened by 14 %. The reduction in the anterior extension was not statistically
1018
ROSEMARY J. JOLLY AND W. J. MOORE
Table 1. Comparison of skulls of normal and en jen mice
Normal
cn/cn
Measurements taken on dried skulls
Overall length of cranium
2-506 ±00203
2037 ±00339
Length of neurocranium
1-684 ±00176
1-360 ±00204
Width of neurocranium
1114 ±00052
1151 ±0045
Height of neurocranium
0-744 ±00090
0-733 ±00079
Width of cranial base
0195 ±00045
0177 ±00057
0-679 ±00160
Length of viscerocranium
0-825 ±00091
Width of viscerocranium
0-515 ±00076
0-517 ±00075
Height of viscerocranium
0-594 ±00079
0-546 ±00064
1-375 ±01772
1-228 ±00208
Length of mandible
0-697 ±00184
Length of condylar process
0-624 ±00088
0-436 ±00155
Volume of endocranial cavity 0-531 ±00096
Length of humerus
1-223 ±00177
0-754 ±00158
Measurements taken on radiographs
10-72 ±0-158
801 ±0-218
Basicranial axis
Anterior extension
5-22 ±0-068
5-30 ±0039
3-77±0-134
Posterior extension
4-37 ±0076
201-0±l-36
206-8 ±1-27
Angle between basicranial
axis and anterior extension
/o
P (t test)
+ .18-8
+ 19-3
-3-3
-1-5
+ 90
+ 17-7
+ 0-3
+ 8-1
+ 10-7
+ 10-5
+ 17-9
+ 38-4
< 0001
< 0001
0-5-0-4
0-4-0-3
005-002
< 0001
0-9-0-8
< 0001
< 0001
001-0001
< 0001
< 0001
+ 24-7
< 0001
0-4-0-3
001-0001
001-0001
+ 1-5
+ 13-7
+ 2-8
Means and standard deviations for linear and angular measurements. Linear measurements in cm, angles in degrees, volume in ml, + indicates value for normal was greater.
Note that radiographic measurements were taken on enlargements. Number of observations
in each group = 1 0 .
significant. The width of the cranial base was 9 % less in the cn/cn than in the
normal animals. The angle between the anterior extension and the basicranial
axis was decreased in the enjen mice by some 3 %. The spheno-occipital synchondrosis did not appear to undergo premature closure, being still open in
the oldest (36 weeks) achondroplasic mouse reared in our colony.
Neurocranium. The length of the neurocranium was reduced by some 19 %
in the cn/cn mice compared to the normal animals. Neither the width nor the
height of the neurocranium showed statistically significant differences between
the two groups.
Viscerocranium. The length of the viscerocranium was shorter by almost 18 %
in the cn/cn mice. The height of the viscerocranium was reduced by just over
8 %. There was no statistically significant difference between the two groups in
the width of the viscerocranium.
Mandible. Both the total length of the mandible and the length of the condylar
process were reduced by almost 11 % in the cn/cn mice compared to the normal
mice.
Humerus. The shortening of the humerus in the cn/cn animals was some 38 %.
Skull growth in mice
1019
DISCUSSION
None of the measurements of the skull in the cn/cn mice was reduced to the
same extent as the length of the humerus, which was taken to provide an indication of the effect of achondroplasia on the growth of epiphyseal cartilage. The
greatest reduction in the cranial dimensions was observed in the basicranial axis
of the cranial base. This finding is not unexpected, since elongation of the
basicranial axis occurs entirely at synchondrotic cartilages (between the sphenoid
and occipital and pre- and post-sphenoid bones) where the processes of endochondral ossification are similar, if not identical, to those occurring in epiphyseal
cartilages (Baume, 1961 a, 1968).-The flattening of the cranial base in the cn/cn
mice, as indicated by the decrease in the angle between the basicranial axis and
its anterior extension, is attributable to the expansion of the growing brain and
meninges within the shortened endocranial cavity. The doming of the skull and
the tendency towards an increase in the height and width of the neurocranium
(the data were insufficient to establish the latter two trends as statistically
significant) may also be attributable to the same effect. As a result, the volume
of the endocranial cavity was reduced less than might have been expected from
the degree of shortening of the neurocranium (allowing for the cube relationship
between volume and linear dimensions).
The extensions of the basicranial axis were reduced by much smaller amounts
than the axis itself. So far as the posterior extension is concerned, this finding
is not surprising in that this component of the skull contains no cartilaginous
growth sites. The anterior extension, however, is coterminous with the endochondrally ossifying mesethmoid. Why this part of the ethmoid complex should
show no statistically significant reduction in length in the achondroplasic animals
is not apparent from craniometric data alone (see below).
The viscerocranium was reduced in both length and height in the cn/cn mice.
Part of this reduction may be due to the diminution in masticatory muscle function secondary to the malocclusion. That this is not the only factor involved is
indicated by comparing the present findings with those of Moore (1967) where
the reduction in viscerocranial growth in rats following bilateral ablation of
the masseter muscles was only some 1-4 %. The much greater reductions in
the cn/cn mice appear to be largely attributable to the chondrodystrophic effect
on the septal cartilage which has been shown in primates (no equivalent information being available for the mouse) to bear a close structural resemblance
to an epiphyseal cartilage in the region of the septo-ethmoidal junction (Baume,
1961 b), there being no reason to assume any diminution in the impetus of sutural
growth except as a secondary effect. The reductions in overall mandibular
length and length of the condylar process were much smaller than those in the
basicranial axis and viscerocranium, and may be more completely attributable
to the diminution of muscle function consequent upon the malocclusion, since
the mandible appears to have a greater susceptibility than the cranium to changes
1020
ROSEMARY J. JOLLY AND W. J. MOORE
in muscle activity - in the rat, bilateral ablation of the masseter muscles led to
a 7 % shortening of the mandible and an 11 % diminution in the length of the
condylar process (Moore, 1967, 1973). The finding that the reduction of condylar growth in the cnjcn mice was much less than the corresponding reductions
in the basicranial axis and viscerocranium supports the suggestion (Griineberg,
1963) that appositional cartilage growth is less affected than interstitial cartilage
growth in achondroplasia.
Organ culture and transplantation studies of the condylar cartilage (Petrovic,
1972) indicate that this structure probably possesses less inherent growth potential than do septal (Petrovic, Charlier & Herrmann, 1968) or epiphyseal and synchondrotic cartilages (Koski & Ronning, 1965, 1966, 1969, 1970). Numerous
extirpative procedures have been performed to determine whether the septal
and condylar cartilages are primary determinants of viscerocranial and mandibular growth respectively, but the results are inconclusive (see, for example,
Moss, Bromberg, In Chul Song & Eisenman, 1968; Sarnat & Muchnic, 1971).
The findings of the present study appear, at first sight, to support the view that
the synchondrotic and septal cartilages, if not the condylar cartilage, are such
primary growth determinants, but an alternative possibility is that their reduced
growth in the en/en mice results in their acting as 'ties' which physically restrain
growth at other sites (see Moore & Lavelle, 1974 for a full discussion of this
problem).
Craniometric data for chondrodystrophies in other mammalian species are
sparse and usually qualitative. The most comprehensive accounts, which include
metrical data, have been given by Stockard (1941), who describes the skull in
dog breeds of disproportionate body form in which the growth defect is believed
to be of a chondrodystrophic nature. He deals most fully with the Bulldog in
which the disproportion is confined to the axial skeleton. Although Stockard's
measurements were not designed specifically to determine the extent of growth
reductions in the various cranial sites of cartilage growth, it is clear that the
deformities in the Bulldog skull are similar to those in the enjen mouse. As compared to a dog of normal proportions (e.g. Alsatian), the cranial base is
shortened, the vault is domed, the upper facial skeleton is reduced in length
and the mandible is also reduced but to a lesser degree than the upper face.
Similar deformities occur in the skulls of the French Bulldog, Boston Terrier,
Pekinese and Brussels Griffon.
. In man, cranial deformities have been described as a constant feature of the
classic form of achondroplasia, helping to distinguish it from other chondrodystrophic conditions such as spondylo-epiphyseal dysplasia (Maroteaux &
Lamy, 1964). The cranial deformities bear many similarities to those just
described in the cnjcn mouse. The cranial base is shortened, the angle between
the basicranial axis and its anterior extension is reduced (to about 90° compared
with a normal value of about 120°), the calvarium is domed and the nose retracted (Maroteaux & Lamy, 1964). The human achondroplasic skull differs
Skull growth in mice
1021
from that in the mouse in that the endocranial cavity is enlarged, but whether
due to megalocephaly or hydrocephaly is uncertain (Dennis, Rosenberg &
Alvord, 1961; Bergstrom, Laurent & Lundberg, 1971), and in the early closure
(or replacement by fibrous tissue) of the spheno-occipital synchondrosis (Benda,
1947). We have been unable to locate any quantitative descriptions of the human
mandible in achondroplasia, but from the numerous accounts of Angle Class III
malocclusions (i.e. prognathous mandibles) in subjects with this condition
(e.g. Weinmann & Sicher, 1955), it appears that the growth of the lower jaw is
affected less than that of the upper jaw, as in the achondroplasic mouse.
An investigation of the growth of the skull of the achondroplasic mouse is
now being carried out using intravital staining of newly formed bone and cartilage. This will enable a direct comparison to be made of (1) the rates of endochondral ossification at the individual cartilaginous growth sites of the cranial
base, to help elucidate the differential effects of this form of chondrodystrophy
on the basicranial axis and its anterior extension, and (2) the rates of ossification at synchondrotic, septal and condylar cartilages.
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(Received 29 November 1974)