/ . Embryol. exp. Morph. Vol. 39, pp. 59-70, 1977
Printed in Great Britain
59
Further studies on skull growth in
achondroplasic (en) mice
By A. K. BREWER, D. R. JOHNSON AND W. J. MOORE 1
From the Department of Anatomy, University of Leeds
SUMMARY
The morphology of the basioccipital, basisphenoid and mandibular bones in achondroplasic (cn/cri) mice was compared with that of normal siblings. The two bones of the cranial
base were markedly reduced in length but not in width. The percentage reduction in the
basisphenoid was twice that in the basioccipital bone and of the same magnitude as that
previously observed in the long bones of the limbs. This difference may arise because the
basisphenoid, like the long bones, grows in length from two cartilaginous growth sites while
the basioccipital grows from one cartilaginous and one periosteal site. The mandible of the
enfen mice was also reduced in size, although to a lesser extent than were the cranial bones
and without the ensuing disproportion. The scale of the mandibular changes suggests that
they are largely attributable to regulatory responses to the shortened cranium. The finding
that the condylar cartilage of the cn\cn mice is reduced in thickness indicates, however, that
the en gene may have a direct effect on condylar chondrocytes.
INTRODUCTION
In a previous paper (Jolly & Moore, 1975), quantitative data were presented
on the growth of the skull in achondroplasic (enjeri) mice. It was shown that
the en gene produces marked changes in skull proportions, in addition to its
well documented effects on the post-cranial skeleton (Lane & Dickie, 1968;
Konyukhov & Paschin, 1970; Silberberg & Lesker, 1975). Particularly prominent
were shortening of the cranial base with corresponding broadening of the calvarium and retraction of the nasal skeleton. From mainly qualitative studies
(e.g. Crew, 1923; Stockard, 1941; Landauer & Chang, 1949; Griineberg, 1963)
of other species, it appears that such cranial changes are common to the
majority of mammalian achondroplasias.
The effects of achondroplasia on the skull are of especial interest in that this
part of the skeleton embraces, as well as the endochondral elements of the
cranial base and sense capsules, the dermal bones of the calvarium and face
which ossify intramembranously and the secondary condylar, angular and
coronoid cartilages of the mandible. The most important of the latter, the
condylar cartilage, has been shown to grow by apposition to its upper surface
as a result of differentiation of chondrocytes in the intermediate cell zone
* Authors' address: Department of Anatomy, School of Medicine, University of Leeds,
LS2 9NL, U.K.
60
A. K. BREWER, D. R. JOHNSON AND W. J. MOORE
(situated between condylar and articular cartilages) rather than the interstitial
proliferation of chondrocytes which predominates at a typical epiphyseal
plate (Blackwood, 1966; Frommer, Monroe, Morehead & Belt, 1968; Silbermann & Frommer, 1972). There is strong evidence that the intermediate cell
zone is of periosteal origin and that the condylar cartilage is, in consequence,
the product of periosteal chondrogenesis (Meikle, 1973). The chondrocytes of
the condylar cartilage do not form columns of hypertrophic cells, as occur in
epiphyseal cartilages, and they emerge, still viable, into the underlying zone of
ossification (Durkin, Irving & Heeley, 1969). In view of these peculiarities, the
question arises whether condylar chondrocytes are affected by the en gene or
whether the effects of this gene are, as suggested by Gruneberg (1963), specific
for epiphyseal chondrocytes.
The aims of the present study are to supplement the work of Jolly & Moore
(1975) by (a) investigating the growth of some individual bones of the skull,
including the mandible, (b) making a developmental study of these bones in
normal mice and their en littermates at a variety of ages and (c) investigating
the feasibility of different techniques for the measurement of skull growth in
the mouse.
MATERIALS AND METHODS
The achondroplasic mice used in this study were re-introduced by one of us
(D.R.J.) from a stock maintained at University College London, the stock
used by Jolly & Moore having become extinct. It should be pointed out that
survival amongst post-weaning cn[cn mice is not good: the results presented
for mice aged 30 days or more may therefore be biased as they are based on
survivors whose phenotype may well be less extreme than the norm.
Vital staining. 16-day-old enjen mice and normal littermates were given an
intraperitoneal injection of 25, 50 or lOOmg/kg bodyweight Procion P8-B
(I.C.I.). A second injection was given 24, 48 or 72 h later, and the animals
killed after a further period of up to 24 h. Heads were skinned, fixed in Bouin's
fluid, decalcified in Gooding & Stewart's decalcifier for 7 days, washed in 5 %
sodium sulphate and running tap water (24 h each), dehydrated and embedded
in wax. Longitudinal sections, 15/*m thick, were washed in 70% alcohol to
remove any remaining picric acid and viewed in incident ultraviolet light.
A second group of animals was injected with 5, 10 or 20 mg/kg tetracycline
(Lederle) in a similar manner, but fixed in 10 % glutaraldehyde, dehydrated in
graded alcohols and embedded in resin after the method of Spurr (1969).
Sections 100 jam thick, were viewed in incident ultraviolet light.
Radiography. Twelve-day-old animals were anaesthetized by intraperitoneal
injection of 60 mg/kg 'Intraval Sodium' (May & Baker) and transported to the
X-ray machine in a warmed insulated container. Antero-posterior views of the
head were taken using an exposure of 0-1 sec on dental fast-apical-bite-plates.
The animals were allowed to recover and the procedure repeated on subsequent
Skull growth in mice
61
Table 1. Animals used in the preparation of papain digestion
Age (days)
Genotype
6
12
16
21
32
42
60
75
100 +
+ /?
cnjcn
5
5
7
7
5
5
5
5
5
5
5
5
5
5
5
5
5
5
days. Measurements were made on enlarged prints taken from the radiographs
(Fig. 1A).
Skeletal preparations. Animals for papain digestion (Table 1) were skinned,
eviscerated and stored at — 20 °C. Flesh was removed by boiling for 5 min,
followed by digestion with a suspension of crude papain at 55 °C for 2-3
days. Bones were bleached in hydrogen peroxide and defatted with acetone.
Selected bones were photographed on a grid of 1 mm2 graph paper. Drawings
were prepared from enlarged photographic prints. Randomly chosen pairs of
bones from normal and cnjcn littermates of the same age were superimposed
for comparison.
RESULTS
The technique of vital staining in hard tissues has been widely used in dentistry
to study the growth of teeth: repeated injections of a suitable dye (e.g. Procion)
which binds to the organic or one (e.g. tetracycline) which binds to the inorganic
portion of bone matrix followed by sectioning and visualization by ultraviolet
light produces a series of well-defined lines of stain; the distances between these
can then be used to measure the rate of growth. This technique has also been
applied successfully to the study of the growth of cortical bone (Tarn, Reed &
Cruickshank, 1974a, b, c; Frost, 1969). There are apparently no studies using
this technique on epiphyseal bone. The reason for this became immediately
apparent when sections of the mandibular condyle were made after two injections of Procion P8-B (Fig. IB). In contrast to the regular lines seen in teeth
the deposition of bone in the condyle is very complex: we were not able to
produce two lines of stain despite variations in dose, interval between injections
or time elapsed between the second injection and fixation.
Daily radiography of normal and abnormal mice has the potential advantage
that the same individual is measured repeatedly, thus decreasing variation. In
fact (Table 2), we found considerable difficulty in obtaining sufficiently accurate
measurements of individual bones from radiographs, and the series was discontinued before completion. We also suspected that a daily general anaesthetic
(necessary because the X-ray facilities at our disposal were situated in the
Dental Hospital, where conscious mice are not encouraged) retarded the growth
of both normal and abnormal mice.
5
EMB
39
62
A. K. BREWER, D. R. JOHNSON AND W. J. MOORE
Fig. 1. (A) Antero-posterior radiograph of 16-day-old cnjcn mouse, showing
measurements taken. (B) Mandibular condyle of 20-day-old normal mouse given two
injections of Procion 8-B at 24-h intervals. x280. (C) Mandibular condylar
cartilage, 16-day-old normal mouse. x56. (D) Mandibular condylar cartilage,
16-day-old cnjcn mouse, x 56.
The use of papain digested material, a traditional and time-honoured method,
therefore proved to be best suited to our requirements.
The mandible (Fig. 2). The mandible of the 6-day-old cnjcn mouse (the
earliest time at which normal and cnjcn sibs could be reliably classified in our
stock (although Lane & Dickie (1968) found classification possible at birth),
was already smaller than that of its normal sib (Fig. 2 A). The overall height
and length were both reduced so that there was little change in the proportions
of the bone (Fig. 5). The normal bone continued to outgrow the abnormal,
Skull growth in mice
63
Table 2. Measurements taken from radiographs of normal and en /en mice
Length of basisphenoid bone
(dimension b)
Nasal spine - intersphenoidal
synchondrosis (dimension a)
Age
A
(days)
+ /?
cn/cn
12
13
14
15
16
20
22
23
28
10-7(1)
11-9 ±0-3 (2)
10-9 ±0-8 (3)
11-5(1)
12-7 ±0-7 (2)
12-0±0-7(3)
12-3(1)
12-5 ±0-3 (3)
12-7(1)
83 (1)
9-5 ±0-2 (2)
8-8 ±0-3 (4)
9-3 (1)
9-9 ±0-6 (2)
95 (1)
9-3 (1)
9-8 ±0-4 (3)
9-7 + 0-5(2)
A
P
+ /?
cn/cn
—
1-1(1)
1-5 ±0-5 (2)
1-1+01 (3)
1-1(1)
1-2 ± 0 1 (2)
11 + 0 1 (3)
1-0(1)
10 ± 0 1 (2)
10 ±0-1 (4)
1-0(1)
10 ±0-2 (2)
1-0(1)
1-0(1)
10 ±0-1 (3)
11 ±0-6 (2)
< 002
< 005
—
< 0-2
—
—
11(0
< 002
1-2 + 0-1 (4)
1-3(1)
—
P
—
< 0-5
< 0-5
—
< 0-5
—
—
< 002
—
Measurements in mm. ± S.E.M.
Figures in parentheses indicate number of animals.
Dimensions a and b refer to Fig. 1 A.
Table 3. Antero-posterior length of the pars basalis of the basioccipital
bone (dimension c, Fig. 3 A)
Genotype
Age
A
f
(days)
+ /?
cn\cn
6
12
16
21
32
60
75
0-95±003 (5)
108 ±0-03 (7)
107 ±004 (5)
1-21 ±0-03 (5)
1-28±006 (5)
1-31 ±006 (5)
1-37±007 (5)
1-36±003 (5)
100 +
1-44±003 (5)
0-84±0006 (5)
0-882±002 (7)
0-876 + 0-01(5)
0-984±002 (5)
0-94 ±004 (5)
1 05±005 (5)
1 01 ±003 (5)
107 ±004 (5)
1-20±003 (5)
42
15-7
100
7-2
291
22-1
15-5
21-9
28-6
241
>
•
P
t
<
<
<
<
<
<
<
<
<
0001
0001
0001
0001
0001
0001
0001
0001
0001
Measurements in mm. ± S.E.M.
Figures in parentheses indicate number of animals.
although towards the end of the period studied the differences became less
marked (Fig. 2G-H).
Basioccipital bone (Fig. 3). From superimposed tracings of the basal portion
of the occipital bone it can be seen that the midline antero-posterior length
(Dimension, c, Fig. 3 A, Table 3) was somewhat decreased in the achondroplasic
mice throughout the period studied. The maximum width of the bone initially
reduced in the achondroplasic mice increased progressively until, at 42 days,
the bone was as wide as that of normal litter-mates.
Basisphenoid bone (Fig. 4). The basisphenoid is made up of a central body,
5-2
64
A. K. BREWER, D. R. JOHNSON AND W. J. MOORE
Fig. 2. Mandibles of normal (thick line) and en]en (thin line) mice aged (A) 6d,
(B) 12d, (C) 16d, (D) 21 d, (E) 32d, (F) 60d, (G) 75d, (H) 159d.
which ossified in cartilage, and two wings which form in membrane. At 6 days,
the antero-posterior length (Dimension d, Fig. 4A, Table 4) and total width
from right to left wings were reduced, but, as with the occipital, the width
increased to that of the normal bone, whilst the length remained subnormal.
Skull growth in mice
65
1 mm
D
Fig. 3. Basioccipital bones of normal (thick line) and cn/cn (thin line) mice aged
(A) 6d, (B) 12d, (C) 16d, (D) 21 d, (E) 32d.
66
A. K. BREWER, D. R. JOHNSON AND W. J. MOORE
H
Fig. 4. Basisphenoids of normal (thick line) and en]en (thin line) mice aged (A) 6d,
(B) 12d, (C) 16d, (D) 21 d, (E) 32d, (F) 42d, (G) 60d, (H) 75d.
Skull growth in mice
67
Table 4. Antero-posterior length of the body of the basisphenoid
(dimension d, Fig. 4 A)
Genotype
Age
(days)
+ /?
cn\cn
t
6
12
16
21
32
42
60
75
0-82 ±001 (5)
1-15±007 (6)
108±003 (5)
118±002 (5)
1-23±007 (5)
1-34±003 (5)
1-32±009 (5)
1-39±005 (5)
1-54±005 (5)
0-63 ±001 (5)
0-71 ± 0 0 2 (7)
0-74±002 (5)
0-80±006 (5)
0-72 ±0-03 (5)
0-91 ±008 (5)
0-92±005 (5)
0-75±003 (5)
1 17 ±011(5)
13-7
38-8
53-4
290
32-2
23-8
180
51-4
15-6
100 +
P
<
<
<
<
<
<
<
<
<
0001
0001
0001
0001
0001
0001
0001
0001
0001
Measurements in mm. ±S.E.M.
Figures in parentheses indicate number of animals.
The wings, which were initially smaller in en, soon achieved parity with normal
litter mates and maintained this condition.
Histology of the mandibular condyle (Fig. 1C, D). The thickness of this
cartilage was much reduced in the abnormal mice but its general structure was
similar in both groups of animals.
DISCUSSION
Cranial base. The areas of cartilaginous growth in the cranial base are three
in number: the spheno-occipital synchondrosis between basioccipital and
basisphenoid, the intersphenoidal synchondrosis between basi- and presphenoids
and the spheno-ethmoidal synchondrosis between the presphenoid and ethmoid.
The 2 5 % decrease in basicranial axis (spheno-ethmoidal synchondrosis to
anterior margin of foramen magnum) length reported by Jolly & Moore (1975)
must be produced by growth reductions in these zones. We have information
on the spheno-occipital and intersphenoidal synchondroses in so far as they
contribute to the basioccipital and basisphenoid bones. The amount of reduction in these bones (Table 5) varies according to the age at which comparison
is made, for the rates of growth of normal and cn\cn bones are not identical.
Because the mice used by Jolly & Moore (1975) were aged 13 weeks or more,
the best comparison is with mice in our study aged 60 and 75 days. The basioccipital in mice of this age group is reduced by about 24% whilst the basisphenoid is reduced by almost twice this amount. The obvious conclusion is
that this difference arises because the occipital has a contribution from one
abnormal growing region, the spheno-occipital synchondrosis (periosteal
osteogenesis at its posterior margin being presumably unaffected by the en
gene), whilst the aboral sphenoid receives contributions from two such regions,
68
A. K. BREWER, D. R. JOHNSON AND W. J. MOORE
Table 5. Percentage reduction in size of cnjcn skull bones
Reduction %
; (days)
6
12
16
21
32
42
60
75
100 +
Basisphenoid
Basi-occipital
14-6
38-2
31-4
23-7
41-4
320
30-3
460
240
11 5
18-3
181
18-6
26-5
19-8
26-2
21-3
15-2
Fig. 5. Mandibles of normal (thick line) and cnjcn (thin line) mice aged 159d. The
cnjcn has been enlarged so that: (A) anterior-posterior length is equal, (B) total
height is equal.
Skull growth in mice
69
the spheno-occipital and intersphenoidal synchondroses. The basisphenoid
may in this respect be compared to a long bone, which also has a growing
region at each end, and indeed the reduction of some 40 % seen in the basisphenoid tallies well with the reduction seen in the humerus (Lane & Dickie,
1968, 34-3%; Jolly & Moore, 1975, 38%; Silberberg & Lesker, 1975, 36%).
The disproportion introduced into the bodies of both basisphenoid and basioccipital of the cnjcn mice resembles that found in the long bones of the postcranial skeleton. In each case, the length of the bone between epiphyses
(synchondroses) is reduced whilst its width remains just below, or indeed in
some cases slightly exceeds, the normal dimension.
Koski & Ronning (1969, 1970) found that synchondritic cartilage grows less
well than epiphyseal cartilage when transplanted, and this has led to a belief
in some quarters that epiphyseal and synchondritic cartilage differ in some
respect. If this is true, then the difference is not reflected in the effects of the en
gene.
Mandible. The mandible, however, presents a rather different picture. Here,
although the overall size is reduced (11 % in Jolly & Moore's study), there is
no sign of disproportion (Fig. 5).
Griineberg (1963) has suggested that appositional growth (as found in the
secondary cartilage of the mandibular condyle) is less affected by achondroplasias than is interstitial growth. The relatively minor morphological effects
observed in the enjen mandible would, by themselves, lead us to go further and
suggest that secondary cartilage is unaffected by this gene, the small reduction
in mandibular size being attributable to regulating changes in response to the
shortened cranial base and the diminution of muscle function consequent upon
the malocclusion that develops in achondroplasic mice (Moore, 1967, 1973).
However, light histology revealed that the cartilage of the mandibular condyle
is reduced in thickness, although not noticeably disrupted, in enjen mice,
indicating that some dysfunction is present. The answer to this problem lies in
the relatively unexplored area of the differences between primary and secondary
cartilage (see Hall, 1975, for a discussion of the possible phylogenetic and
ontogenetic significance of these tissues). Whatever these differences might be
(and we are a long way from finding out, as ultrastructural studies of mandibular secondary cartilage are just appearing - Silbermann & Frommer, 1972;
Appleton, 1975), it seems possible that the genetically determined achondroplasias might prove a useful investigatory tool. They are clearly multifactorial
in origin (Rimoin, 1975) and it may well be that one or other will be found to
affect the mandibular condyle differentially.
70
A. K. BREWER, D. R. JOHNSON AND W. J. MOORE
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