J. Embryol. exp. Morph. Vol. 38, pp. 227-238, 1977
Printed in Great Britain
227
Mandibular growth
retardation as a cause of cleft palate in mice
homozygous for the chondrodysplasia gene
By ROBERT E. SEEGMILLER 1 AND F.CLARKE FRASER
From the Department of Zoology, Brigham Young University, Provo, Utah,
and the Department of Biology, McGill University, Montreal
SUMMARY
Defective chondrogenesis in C57BL mice homozygous for the chondrodysplasia gene leads
to deformity of limbs, ribs, trachea, mandible and palate. Since formation of the secondary
palate depends upon coordinated development of several craniofacial structures, the presence
of micrognathia and cleft palate in cho/cho newborn mice suggested a cause-and-effect
relation between these two deformities. To determine whether or not lower jaw shortening
coincided with the time of palate closure, heads from mutant and control littermates previously
rated morphologically were examined in median sagittal section. Of six parameters analyzed,
growth rates for mutant mandible and anterior vertical dimension were significantly less than
those of controls from the beginning of control palate closure. Since there is evidence that intrinsic shelf force is normal, these observations suggested that, during palatogenesis, growth
retardation of Meckel's cartilage did not allow forward displacement of the tongue, and that
the consequent failure to straighten the tongue impaired shelf movement. The data support
the concept that growth of Meckel's cartilage is necessary for normal palate formation.
INTRODUCTION
Although the precise role played by the lower jaw in development of the secondary palate is not known, evidence is increasing that mandibular growth
retardation during embryogenesis can be a contributing factor to cleft palate
(Cohlan, 1953; Thalhammer & Heller-Szollosy, 1955; Asling et al. 1960;
Fitch, 1961; Latham, 1965; Schwarz & Chaudry, 1968; Fraser, 1969; Burdi
et al. 1972; Ohyama, Pruzansky, Heinze & Parris (1974). A causal relation,
however, cannot be determined by the mere association at birth of micrognathia
and cleft palate but only by demonstrating that mandibular growth retardation coincides with palate development. Indeed, Shih, Trasler & Fraser (1974)
have shown that a teratogen (vitamin A) that causes micrognathia and cleft
palate, causes the micrognathia after palate closure in the control is complete, so
1
Author's address: Department of Zoology, Brigham Young University, Provo, Utah
84602, U.S.A.
228
R. E. S E E G M I L L E R
A N D F. C.
FRASER
that the micrognathia does not appear to cause the cleft palate in this case.
However, this may not apply to all cases of cleft palate with micrognathia.
Mice with completely penetrant, recessive chondrodysplasia {chojcho)
(Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson & Sheldon, 1972) are
born with micrognathia and cleft palate and are therefore a suitable model for
study of the role of the lower jaw in palate formation. From sagittal measure­
ments of craniofacial structures associated with palate closure, this study suggests
that mandibular growth retardation resulting from defective chondrogenesis is
the principal cause of cleft palate in cho mice.
MATERIALS
AND
METHODS
Mature heterozygous female mice {choj ±) of the C57/BL/Fr strain were
placed overnight with heterozygous males. The presence of a copulation plug
the following morning indicated that mating had occurred and that the embryos
were approximately 6 h old (day 0/6, Snell, Fekete, Hummel & Law, 1940).
Between days 13/20 and 15/4, pregnant uteri were excised and preserved in
Bouin's fixative. After fixation for 48-72 h the fetuses were removed from the
uterus and rated morphologically (MR) according to Walker & Crain's method
(1960). The heads were removed and further processed for examination by light
microscopy. Sagittal sections were cut at 10 //m and stained with toluidine
blue. Mutants were easily identified by reduced staining intensity of the cartilage
matrix (Seegmiller et al. 1971). Midsagittal sections showing thyroglossal duct
and nasal septum were photographed at low magnification using a Zeiss photomicroscope with a plan 1 x objective. The overall enlargement from tissue to
print was approximately 35 x . Reference points were marked on acetate matte
overlays (Fig. 1) and measurements of the head were made in millimeters or
degrees by Hart, Smiley & Dixon's method (1972). These measurements per­
mitted correlation of palate closure stage (PS 0-7, Trasler, 1965) with the fol­
lowing parameters: maxillary and mandibular growth, changing angulation of
the cranial base and lower jaw, and changing vertical dimension of anterior and
posterior regions of the mouth. Palate closure stage was determined by examin­
ing serial sagittal sections for the position of the shelves.
Most fetuses were collected over a period of 2 years. Twenty-five litters were
examined, from which most morphological stages from M R 0-15 were repre­
sented. The number of controls recovered was 90; the number of mutants was
19, which approached the 25 % expected frequency for a recessive gene. Because
the MR of the 19 mutants was random, there were stages which consisted of few
or no mutants. For statistical purpose, morphological stages were therefore
grouped, such that each group was represented by at least two mutant fetuses
(Table 1). Mutant and control littermates generally were of the same group. A
comparison of the curves before and after grouping suggested that this procedure
did not affect the shape of the curves. Differences were tested by the M a n n -
Chondrodysplasia, micrognathia and cleft palate in mouse
229
Anterior
vertical
Fig. 1. Median sagittal section of a day-14 (MR 4) control fetal head showing
position of tongue and mandible prior to palate closure. Labels indicate the parameters measured in millimeters or degrees. Enlarged approximately 35 x .
Whitney U test (Siegel, 1956); a probability of less than 5 % was considered
significant.
RESULTS
Palate closure stage vs. morphological age
Palate shelves of control fetuses at MR 0-3 were vertically orientated (PS 0). At
MR 4 and 5 the shelves were either vertical (PS 0) or in the early stages of shelf
movement (PS 1-3). Between MR 6 and 11 the shelves were generally horizontal
(PS 4) and many were fusing (PS 5-6). At MR 13-15 closure of the secondary
palate was complete (stage 7). Timing of palate closure was also plotted against
chronological age. Fetuses recovered late on day 13 or early on day 14 generally
had open palates; those recovered between days 14/6 and 14/12 were at intermediate stages of palate closure; and those recovered late on day 14 or early on
day 15 had closed palates. Palate closure time for control C57BL mice was similar
to that reported by Trasler (1965) and Shih et al. (1974), although the shape of the
curve deviated slightly, perhaps due to our use of tissue sections rather than
whole heads to determine palate stage.
Mutant fetuses were randomly distributed from MR 0-15. A comparison of
mean MR for control and cho mice within litters suggested that the cho gene
did not affect the MR. Mutant heads examined in sagittal section consistently
showed palate shelves in the vertical position. Although there was no evidence
of movement to the horizontal position, the shelves were capable of moving
Control
cho
cho, % control
Control
cho
cho, % control
Control
cho
cho, % control
Control
cho
cho, % control
Control
cho
cho, % control
0-2
14
4
7
5
17
4
33
2
19
4
N
94-1 ±2-2
91-8 + 5-3
97-6
112-6± 1-2
107-5 ±6-6
95-5
114-1 + 1-6
113-5 + 5-6
99-5
123-0 ±2-9
120-6 ±4-7
980
142-8 ±2-2
141-2 ±6-5
98-9
(mm)
151-0± 11
149-0 ±2-7
98-7
159-5±0-7
156-0 ±2-0
97-8
159-8±l-0
154-5 ±5-3
96-6
163-7 ±0-8
163-6±l-8
99-9
169-8 ±1-7
168-5+1-2
99-2
3C±S.E.
Cranial base
(degrees)
52-6±ll
51-8±2-4
98-5
67-0±l-0
580±41
86-6*
69-5 ±1-2
61-2 + 3-3
88-1*
79-9±l-7
68-0 + 2-0
851
94-8 ±2-4
78-8 + 1-1
83-1J
Mandibular
(mm)
5-5±O-8
4-5±l-3
81-8
16-0±0-7
8-5±2-5
53-1*
19-7±l-0
12-2 ±2-0
61-9*
23-6 ±0-6
14-0±2-3
59-3*
31-1 ± 1-3
19-5 ±0-4
62-7J
Anterior vert.
(mm)
21-8±0-4
220 ±0-7
100-9
22-9 ±0-5
21-8± 1-5
95-2
24-3 ±0-5
22-2 ±0-6
91-4
22-4 ±0-6
21-l±0-9
94-2
26-6 ±1-5
29-8 ±0-8
1120
gy
py
,
y
y
t Two to four morphological groups were combined in order to increase the sample size. Total number of control fetuses equals 90, mutants
equals 19.
% Control and cho means differed significantly at the 001 probability level.
38-1 ±0-6
37-5±2-4
98-4
38-0±0-9
360 ± 2 0
94-7
40-3 ±0-8
41-5±7-0
1030
37-6 ±0-7
32-6 ±1-7
86-7*
38-6±l-5
38-0 + 3-2
98-4
Posterior vert. Posterior angle
(degrees)
(mm)
* Control and rhn means differ*;d sienifn=antlv at the. 005 nrohahilitv level determined hv tii e Mann—Whitne/v TT test
13-15
8-11
5-7
3-4
Group
MRf
Table 1. Craniofacial dimensions {taken from x 35 prints) for normal and cho fetuses during palatogenesis
m
1*
p
m
&
r
>—i
O
W
in
w
Chondrodysplasia, micrognathia and cleft palate in mouse
231
Mandibular length
100
Control
A
0-66
A Mutant
90
0-56
A
80
Mand./max. .
ratio
/
-
t
70
/
o-56 y
0-2
3-4
5-7
Morphological rating
8-11
13-15
Fig. 2. Mutant mandibles were significantly shorter throughout palate closure.
The mandibular/maxillary ratio for mutants did not increase as it did in the
control.
towards the horizontal when the lower jaw and tongue of two mid-day 14
mutant fetuses were mechanically depressed. Furthermore, the palates of two
other mutants, born with spontaneous agnathia and microglossia, were closed.
Maxillary length
Maxillary length for control fetuses increased from 94-1 mm at MR 0-2 to
142-8 mm at MR 13-15 (Table 1). The increase in mutant maxillary dimension
closely paralleled that of the controls and at no point did the value for the
mutant differ significantly from that of the control.
Cranial base angle
The cranial base angle for control fetuses increased from 151-0° at MR 0-2 to
169-8° at MR 13-15 (Table 1). The rate of increase in angulation for mutant
cranial base did not differ significantly from that of the control (Table 1).
232
R. E. SEEGMILLER AND F. C. FRASER
Fig. 3. (A) Day-15 (MR 15) control midsagittal section showing horizontal shelf,
relatively straight tongue, and a line extending through hyoid cartilage, Meckel's
cartilage and apex of lower jaw. Force from primary palate is directed through the
anterior lower jaw (arrow). Distance between hyoid and Meckel's cartilages is
indicated (50 mm is representative of MR 15 fetuses). (B) Day-15 (MR 15) mutant
midsagittal section of cleft palate fetus showing curvature of tongue, and shortening
of mandible as measured from hyoid to Meckel's cartilage (35 mm, representative). A
line extended through hyoid and Meckel's cartilages traverses above the apex of the
lower jaw, through the tongue and maxilla. The line of force (arrow) from the primary
palate is directed through the tip of the tongue but anterior to the lower jaw.
Mandible length
Mandibular dimension for control fetuses increased from 52-6 mm at MR 0-2
to 94-8 mm at MR 13-15 (Table 1, Fig. 2), an increase of approximately 1-8
times. Mutant mandibular dimension increased from 51-8 mm at MR 0-2 to
Chondrodysplasia, micrognathia and cleft palate in mouse
233
78-8 mm at MR 13-15 (Table 1), an increase of approximately 1-5 times. Mandibular shortening was significant and present as early as MR 3-4 (86-6 % of
control, Table 1), during the commencement of palate closure. At MR 13-15,
i.e. after palate closure, mutant mandibular dimension was 83-1 % of control.
Thus throughout palate closure, mutant lower jaws were consistently and significantly shorter than those of the controls (Fig. 2). However, the mandible anterior
to Meckel's cartilage, which is comprised of soft tissue, was not reduced in
length, and the distance between Meckel's cartilage and the hyoid cartilage was
only 70 % of the control measurement (Fig. 3 A, B). This suggests that the reduction in mandibular length occurs primarily through retardation in growth of
Meckel's cartilage.
Mandibular growth retardation was further demonstrated by comparing
mutant and control for mandibular/maxillary ratio (Fig. 2). Whereas the ratio
for control increased from 0-56 at MR 0-2 to 0-66 at MR 13-15, the ratio for
cho fetuses was approximately 0-56 throughout palate closure.
Anterior vertical dimension
This parameter represents the degree to which the mandible rotates as the
mouth opens. For control fetuses this measurement increased from 5-5 mm at
MR 0-2 to 31-1 mm at MR 13-15 (Table 1). Mutant anterior vertical dimension increased from 4-5 mm at MR 0-2 to 19-5 mm at MR 13-15. The difference
between mutant and control at MR 0-2 was not statistically significant whereas
the difference thereafter was (Table 1). The greatest difference was observed at
MR 3-4 when mutant vertical dimension was 53-1 % of control.
Posterior vertical dimension
This parameter for control fetuses ranged from 21 -8 mm at MR 0-2 to 26-6 mm
at MR 13-15 (Table 1). Mutant posterior vertical dimension increased similarly
from 22-0 mm at MR 0 to 29-8 mm at MR 15. At all stages the mutant did not
differ significantly from control for posterior vertical dimension.
Posterior angle
This dimension for control fetuses was 38-1 at MR 0-2 and 38-6 at MR 13-15
(Table 1) and for cho fetuses it was 37-5 at MR 0-2 and 38-0 at MR 13-15. The
deviation from control was significant only at MR 8-11.
DISCUSSION
During the time of active chondrogenesis in normal C57BL fetuses the palatine
shelves reorientate themselves from vertical to horizontal, and fuse above the
tongue to form the secondary palate. The mechanism for shelf movement is not
completely understood. It may involve, among other things, extension of the
cartilaginous cranial base (Harris, 1964; Verrusio, 1970; Long, Larsson &
Lohmander, 1973) and contraction of actin-like proteins within the shelves
234
R. E. SEEGMILLER AND F. C. FRASER
(Lessard, Wee & Zimmerman, 1974; Babiarz, Allenspach & Zimmerman, 1975;
Wee, Wolfson & Zimmerman, 1976).
Evidence from the present study suggested that shelf force in cho fetuses is
normal. The mutant's shelves before closure appeared histologically and
morphologically normal, and upon surgical removal of the tongue and lower
jaw they rotated horizontally. They were also fused in cho fetuses born
with spontaneous agnathia and microglossia. These observations suggested
that the underlying cause of cleft palate in cho fetuses was not related to intrinsic shelf force,
Before palate closure the tongue lies between the shelves and behind the
primary palate. Afterwards the tongue lies below the shelves, which have become
horizontal, and below the primary palate. Although the evidence is controversial (see Larsson (1974) for review), tongue displacement seems necessary for
shelf rotation and probably involves tongue-primary-palate contact (Fraser,
1968) and possibly mouth opening reflexes (Walker, 1969; Walker & Ross, 1972).
The lower jaw has been associated with tongue displacement because of its
attachment to the tongue and because both undergo rapid growth during the
time of shelf rotation (Sicher, 1915; Zeiler, Weinstein & Gibson, 1964; Hart,
Smiley & Dixon, 1969; Burdi & Silvey, 1969; Wragg, Klein, Steinvorth &
Warpeha, 1970; Diewert, 1974). In the present study the lower jaw of control
fetuses nearly doubled its length between MR 0 and 15 (Fig. 2), stages which
immediately precede and follow palate closure.
Further support for the developmental relation between jaw growth, tongue
displacement and palate closure comes from several examples of cleft palate that
are associated with mandibular shortening or ankylosis (Sisken, GluecksohnWaelsch, 1959; Deuschel & Kalter, 1962; Latham, 1965; Nanda, 1970; Ohyama
et al. 1974). Burdi et al. (1972) observed that ' . . . a majority of syndromes in
which micrognathia is a feature also include cleft palate as a feature, whereas
many syndromes involving cleft palate do not include micrognathia. This
provides indirect evidence that micrognathia of Pierre Robin [and of cho]
syndrome is a contributory cause to the cleft palate'.
The cho mandibular dimension was 98-5 % of control prior to shelf rotation
(MR 0-2), 86-6 % during the initial stages of palate closure (MR 3-4) and 83-1 %
after closure (MR 13-15, Table 1). Since the average distance between hyoid
and Meckel's cartilage for cho fetuses at MR 13-15 was 70 % of control (Fig. 3),
and since the mandibular skeleton during palate closure is comprised predominantly of well-developed cartilage (extensive intramembranous bone formation
has yet to occur), defective chondrogenesis alone apparently accounted for
mandibular growth retardation.
Growth retardation was further demonstrated by comparing mutant and
control fetuses for mandibular maxillary ratio. The ratio for control increased
from 0-56 at 0-2 to 0-66 at MR 13-15 (Fig. 2). A similar increase, to the point
of transient prognathism, was reported during palate closure in lower animals
Chondrodysplasia, micrognathia and cleft palate in mouse
235
and human beings (Polzl, 1905; Sicher, 1915; Zeiler, Weinstein & Gibson,
1964; Wragg et al 1970; and Die wart, 1974), and was interpreted as accelerated
growth of the lower jaw. The ratio for cho fetuses at MR 0-2 was also 0-56 but
no increase during palate closure was observed. Although the mutant's mandibular dimension showed an absolute increase from 51-8 mm at MR 0-2 to
78-8 mm at MR 13-15 (Table 1), the absence of an increase relative to maxillary
dimension can be interpreted as no acceleration of growth during the critical
time for palate closure.
Electron microscopy provided evidence that the cho gene affects only chondrogenic tissues (Seegmiller et al. 1971, 1972). The presence of large collagen fibrils
in the matrix of Meckel's cartilage (unpublished) confirmed that cartilage of the
lower jaw was also defective. This supports the hypothesis that defective chondrogenesis of Meckel's cartilage causes mandibular growth retardation. The
absence of statistically significant deviations from control for cranial base angle
and maxillary dimension (Table 1) suggested that mandibular growth retardation
alone, acting through the tongue, causes cleft palate in cho fetuses.
The diminished anterior vertical dimension of mutant fetuses (Table 1) present
at MR 3-4, and the (underlying) distortion of the tongue and mandible during
palate closure suggest a mechanism by which the mandibular growth deficiency
causes cleft palate. A line drawn through the control lower jaw at the beginning
of palate closure extends from the posterior cranial base through hyoid and
Meckel's cartilage and the apex of the lower jaw (Fig. 1). The tongue is only
slightly curved and positioned posterior to the primary palate. After palate
closure the tongue is relatively straight and lies below the primary and secondary palate. Despite the morphological changes in the tongue and secondary
palate, a line traversing the lower jaw passed through both cartilages and
apex (Fig. 3 A), as it did before closure. In the mutant, prior to palate closure,
this line extended similarly through all structures of the lower jaw. However,
after the normal closure time it did not extend through the apex of the lower jaw
but above it, traversing the tongue and primary palate (Fig. 3B). The tongue
remained curved and between the vertical palate shelves. It is suggested that, in
the mutant when the tongue is not carried forward by the extension of Meckel's
cartilage, and does not come to lie under the primary palate, it is pressed up
against the anterior part of the cranial base and adopts its curvature. Its tip therefore points downward instead of anteriorly, and depresses the anterior part of
the lower jaw which, anterior to Meckel's cartilage, is still only soft tissue. This
would result in the body of the tongue being arched upwards into the space
between the shelves, which would increase the obstruction of the tongue to shelf
movement so much that they would not reach the horizontal position in time to
fuse.
The above suggests that accelerated growth of Meckel's cartilage plays a
critical role in palate formation; the anterior tip of the cartilage, moving anteriorly, may bring the tongue to lie under the primary palate. This would permit
236
R. E. SEEGMILLER AND F. C. FRASER
the tongue to be straightened and cause its tip to lie between the primary palate
and tip and the lower jaw. Thus, the entire lower jaw would be depressed in
a hinge-like fashion, thereby increasing the anterior vertical dimension, and
creating space for the shelves to move into (Eraser, 1969).
The concept that Meeker s cartilage enhances growth of the lower jaw and
thereby plays a special role in palate development is further supported by the
uniqueness of mandibular ossification. Meckel's cartilage forms approximately
1 day before palate closure (Bhaskar, 1953). Not until after palate closure and
formation of Meckel's cartilage does significant ossification occur, and then it
occurs lateral to the cartilage. The mandible is not an endochondrial bone and
therefore does not require Meckel's cartilage for its formation, although Meckel's
cartilage may contribute to mandibular shaping (Hamilton, Boyd & Mossman,
1964). This suggests that Meckel's cartilage functions during the critical stages
of palate closure to provide a point of attachment to the tongue, and to lengthen
and provide rigidity to the lower jaw.
Our current understanding of the cho gene is that through defective collagenproteoglycan interaction (Stephens & Seegmiller, 1976) it expresses itself only in
hyaline cartilage causing interstitial growth retardation and loss of matrix
rigidity. Growth of the lower jaw is retarded primarily because of the presence of
Meckel's cartilage. This study supports the concept that mandibular growth
retardation during palate closure prevents tongue displacement necessary for
shelf movement and is therefore the primary cause of cleft palate in the cho
mutant.
This research was supported by a Basil O'Connor Starter Research Grant from the National
Foundation - March of Dimes and by the Medical Research Council of Canada. Technical
assistance of Vaunda Barrus and Devon Hiatt is gratefully acknowledged.
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(Received 14 September 1976)