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Frontonasal dysplasia in 3H1 BrBr mice.

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Frontonasal Dysplasia in
3H1 Br/Br Mice
Department of Anthropology, Harvard University, Cambridge, Massachusetts
Graduate Program in Cell and Molecular Biology, University of Hawaii
School of Medicine, Honolulu, Hawaii
Laboratory of Immunology, National Cancer Institute, Frederick, Maryland
Pacific Biotechnology Research Center, Department of Anatomy and Reproductive
Biology, University of Hawaii School of Medicine, Honolulu, Hawaii
Department of Anatomy and Reproductive Biology, University of Hawaii
School of Medicine, Honolulu, Hawaii
The adult Brachyrrhine (3H1 Br/⫹) mouse displays severe midfacial retrognathia, with a “pugnose” external appearance, but information concerning
craniofacial morphology of the homozygote (3H1 Br/Br) mutant is lacking. This
study characterized craniofacial phenotype and genotypic features of the homozygous condition. Segregation analysis was performed by phenotypic scoring of offspring from 3H1 Br/⫹ reciprocal matings. Whole-mount staining was
undertaken to determine the presence or absence of cranial base structures in
newborn and adult mice, while features of cranial base chondrification were
examined using light microscopy and type II collagen immunohistochemistry.
Karyotype analysis was performed to determine whether gross chromosomal
aberrations were present. Finally, microsatellite mapping analysis was undertaken to provide further resolution of the Br locus. Results showed that Br was
inherited as an autosomal semidominant feature. 3H1 Br/Br mice consistently
lacked a presphenoid (with its lateral projections, including a preoptic root,
postoptic root, and lesser wing). Karyotyping did not reveal major gross aberrations; however, microsatellite analysis localized Br to distal mouse chromosome 17 in the vicinity of D17Mit155. These results indicated that 3H1 Br/Br
mice show characteristic features of frontonasal dysplasia, including median
facial clefting and bifid cranium, as well sphenoidal malformations. Furthermore, this mutant should serve as a useful model for examining mechanisms
of frontonasal dysplasia. Anat Rec Part A 271A:291–302, 2003.
2003 Wiley-Liss, Inc.
Key words: median facial cleft; presphenoid; Br; mutation
The adult Brachyrrhine mouse mutant (3H1 Br) has
been reported to exhibit severe maxillary retrognathia
with a “pugnose” external craniofacial morphology (Lozanoff, 1993, 1999) (Fig. 1). In previous morphological studies of postnatal mutants (Lozanoff et al., 1994; Ma and
Lozanoff, 1996), a primary lesion was found in the presphenoid and sphenoethmoidal regions of the anterior cranial base. In vivo and in vitro experiments suggested that
chondrocytic proliferation was reduced in these cranial
base regions (Ma and Lozanoff, 1999, 2002; Lozanoff,
1999). In a subsequent study regarding offspring of reciprocal 3H1 Br/⫹ ⫻ Br/⫹ matings (Singh et al., 1998), three
discrete craniofacial morphologies were noted: 1) normal
craniofacial appearance, 2) pugnose external appearance,
and 3) craniofacial configuration characterized by a deep
median facial cleft. These three morphologies were tentatively identified as 3H1 ⫹/⫹, Br/⫹, and Br/Br. However,
Grant sponsor: Medical Research Council; Grant number: 1069;
Grant sponsor: Hawaii Community Foundation; Grant number:
20012653; Grant sponsor: Sigma Xi.
*Correspondence to: Scott Lozanoff, Ph.D., Department of
Anatomy and Reproductive Biology, University of Hawaii School
of Medicine, Honolulu, HI 96822. Fax: (808) 956-9481.
Received 30 May 2002; Accepted 11 November 2002
DOI 10.1002/ar.a.10034
Published online 7 March 2003 in Wiley InterScience
Fig. 1. Adult (A and B) 3H1 ⫹/⫹ and (C and D) Br/⫹ crania viewed from the lateral perspective. The 3H1
Br/⫹ mice display a cranial cavity (cc) similar in form to normal mice, but the mutants show severe
retrognathia of the midface (m). The 3H1 Br/Br mice do not survive beyond birth. Bar ⫽ 10.0 mm.
the craniofacial morphology of the 3H1 Br/Br homozygous
condition has never been systematically described. The
purpose of this study was to characterize the craniofacial
morphology of the 3H1 Br/Br mutant mouse, with special
emphasis on cranial base development, while also providing additional resolution of the Br locus.
taken. Offspring were removed via Caesarian section between E16 –E18 (31 litters) and scored for the three
craniofacial morphologies (normal, midfacial hypoplasia,
or median facial cleft), as previously described (Singh et
al., 1998; Diewert and Lozanoff, 2002). A ␹2 test was used
to determine whether the incidence of Br differed significantly from a 1:2:1 ratio.
Whole-Mount Staining
All procedures were carried out in accordance with
IACUC specifications, and were approved by the Laboratory of Animal Services, University of Hawaii. Adult 3H1
Br mice were housed under standard conditions with a
12-hr light cycle, and supplied with tap water and Purina
Mouse Chow ad libitum. Embryos were obtained through
reciprocal crosses between Br adults. Females were examined for a vaginal plug starting at 8:00 am and checked
hourly over a 12-hr period. If none was present, the females were removed and remated the next day. The day
on which a vaginal plug was observed was designated as
E0. For immunohistochemical studies, prenatal mice were
obtained prior to extensive ossification of the cranial base.
Offspring from reciprocal matings were born with severe
median facial clefts and died within 24 hr following birth;
therefore, mutants demonstrating median facial clefts included only prenatal or newborn animals.
Whole mounts of crania were prepared using a modification of the technique described by Inouye (1976). Briefly,
crania were removed, brain tissue was extracted, and
heads were fixed in 95% ethanol. Cranial cartilage was
stained with 0.3% alcian blue (8GX). Specimens were then
dehydrated in 95% ethanol, counterstained with 0.1% alizarin red, and macerated with KOH. Specimens were
cleared with increasing concentrations of glycerol. The
sample consisted of 25 newborn mice and 33 postnatal
mice at least 17 days of age. Each mouse cranium was
observed with a Leitz dissection scope at 10⫻ and 30⫻
magnification, and cranial base cartilages and ossification
centers were observed for their presence or absence. When
present, features were qualitatively scored as normal or
Segregation of Br
To test the inheritance pattern of Br, reciprocal matings
between inbred parents (F7-10 generations) were under-
Histological features of the dysmorphic cranial base in
3H1 Br/⫹ and Br/Br animals were assessed using light
microscopy and type II collagen immunohistochemistry
and compared to the normal condition. Pregnant females
were killed by cervical dislocation, and embryos (E17)
were removed from the uterus and rinsed in PBS (pH 7.4).
The heads were removed and retained for transverse sectioning. Chucks were suspended in isopentene and frozen
in liquid nitrogen, and tissues were mounted. Tissue
blocks were positioned in a cryostat and serial sections
were cut at 8 ␮m thickness. Sequential sections were
placed on five different gelatinized slides, with four sections on each slide, and air-dried for 1 hr. The sequential
sections were stained with either toluidine blue or hematoxylin and eosin (H&E) for anatomical reference. Additional specimens were collected for paraffin-embedding
and sectioning. Specimens were collected (as above) and
fixed in 10% neutral buffered formalin for at least 1 week.
Specimens were dehydrated in a graded series of ethanol
(50%, 70%, 95%, and 100%) and cut in a transverse or
sagittal plane at 10 ␮m thickness. Sections were stained
with H&E.
Type II collagen staining was achieved by fixing tissues
with methanol (–20°C for 10 min). The sections were
washed twice (5 min each) with PBST (PBS and 0.05%
Tween 20) at room temperature. E9 primary antibody
(1:100; gift of Dr. E. Craemer, University of Tennessee)
was applied. The sections were then washed with PBST,
and FITC-conjugated goat-anti-mouse Fab fragment was
applied (1:100, 45 min). Slides with tissue sections were
incubated in a moist chamber for 30 min at room temperature, washed twice in PBST (5 min each), and coverslipped using Citifluor mounting medium (Marivac, St.
Laurent, QC). The tissues were viewed and photographed
using a Zeiss fluorescence microscope equipped with a
digital camera. Two sets of negative controls were processed in a similar fashion. In one set, PBS was substituted for the primary antibody, while in the second set
same-species antisera were substituted for the primary
antibody. No qualitatively significant staining appeared
on negative controls.
Karyotype Analysis
Karotypes were generated from normal mice and offspring displaying median facial clefts (n ⫽ 5 each) to
assess the occurrence of gross chromosomal abnormalities. Lymphocytes were used to establish the chromosomal
complement for each animal. The liver was removed,
placed in Hank’s solution, trypsinized (2 min), incubated
with fetal bovine serum, and centrifuged. The supernatant was removed and the pellet was resuspended in modified Eagle’s medium (MEM) (37°C for 2.5 hr). Colcemid
(0.005 ␮g/ml) was added to each culture for the final hour.
Cells were harvested by centrifuging the culture, removing the supernatant, and adding 0.5M KCl for 30 min. The
suspension was centrifuged, and the supernatant was
then removed and fixed in methanol/acetic acid. Final
centrifugation was followed by resuspension in 0.5 ml of
fresh fixative. Two drops of cell suspension were placed
onto a clean slide in a humidified room and air-dried.
Giemsa staining was used to reveal a G-band pattern, and
the chromosomes were photographed and karyotyped.
Metaphases were evaluated for chromosome number,
complement, and gross aberrations.
Microsatellite Linkage Analysis
An interspecific reciprocal backcross was conducted
using 3H1 Br males and Mus castaneus females. Mice
were scored at weaning. They were then killed, and
DNA was extracted from the whole bodies (n ⫽ 93–108).
Based on previous findings by Beechey et al. (1997),
seven microsatellite markers on chromosome 17 were
utilized: D17Mit128, D17Mit122, D17Mit155, D17Mit189,
D17Mit190, D17Mit221, and D17Mit123. Microsatellite
primers (Research Genetics, Huntsville, AL) were synthesized based on sequences listed at http://www.informatics. One oligonucleotide of each pair was labeled
using ␥-[32-P]-ATP and T4 polynucleotide kinase. Pairs of
labeled and unlabeled oligonucleotides were used to amplify each marker using a Perkin Elmer (Foster City, CA)
9600 thermocycler with a PCR profile consisting of 35
cycles for 1 min at 94°C (denaturation), 30 sec at 57°C
(annealing), and 2 min at 72°C (extension). The 32P-labeled PCR products were separated by electrophoresis in
6% denaturing polyacrylamide gels. Gels were dried and
exposed to Kodak x-ray film, and the genotypes were
scored. As an initial analysis, DNA samples from four 3H1
⫹/⫹ and four Br/Br mice were tested to ensure that all
microsatellites amplified and that none were absent (e.g.,
as a result of a megabase deletion in the Br locus). All
microsatellites amplified in all samples. Then, all microsatellites were scored for backcross progeny. Data were
analyzed with Map Manager software (http://mcbio. The Map Manager output
was used to identify likely data errors (apparent close
double recombinants). These were excluded from the analysis and the process was repeated. A two-point LOD score
analysis was conducted between all pairs of markers, and
the probable position of the Br locus was calculated relative to surrounding microsatellite markers.
Segregation Analysis
Segregation analysis showed that offspring separated
into three groups based on craniofacial morphology (Figs.
1 and 2). Of 116 fetuses, 34 were scored as ⫹/⫹, 51 as
Br/⫹, and 31 as Br/Br. A ␹2 value of 1.37 (d.f. ⫽ 2; P ⬍
0.45) was calculated, indicating that the observed proportions were not significantly different from those expected.
Therefore the null hypothesis was accepted, indicating
that the three craniofacial morphologies did not differ
from the expected 1:2:1 ratio (⫹/⫹:Br/⫹:Br/Br).
Gross Morphological Features of the Murine
Cranial Base
Midline cranial base structures in mice include the basioccipital, basisphenoid, presphenoid, and nasal septal
cartilage (Fig. 3). In normal mice, the basioccipital and
central bodies of the basisphenoid and presphenoid mature endochondrally in a caudo-rostral direction. The most
posterior bone, the basioccipital, has a single ossification
center in the mouse, and is homologous to the basilar
process in humans. The posterior border of the basioccipital frames the anterior portion of the foramen magnum,
while the anterior border of the basioccipital forms the
spheno-occipital synchondrosis with the basisphenoid.
The basisphenoid is composed of a central body and a pair
of lateral projections, or greater wings (ala temporalis),
which are homologous to the posterior body of the sphenoid (the clivus and most of the hypophyseal fossa) and
the greater wings in humans. The pituitary sits between
the basisphenoid body and spheno-occipital synchondrosis
Fig. 2. Typical facial morphology of a 3H1 Br/Br mouse shows (A) midfacial retrognathia with (B) a deep
median facial cleft (arrow). Whole-mount staining shows a reduced nasal capsule (ⴱ) in (C) 3H1 Br/Br mice
as compared to (D) normal mice (nc). Bar ⫽ 2.0 mm.
in a central location, thus the hypophyseal fossa in mice
comprises only a slight indentation in the body. The three
components of the basisphenoid originate from separate
growth centers that eventually fuse. The anterior border
of the basisphenoid forms the presphenoidal synchondrosis with the posterior border of the presphenoid. The presphenoid ossifies from a central body, and lateral projections, the lesser wings (orbitosphenoids), ossify
progressively from the central body outward. In addition,
the preoptic and postoptic roots form as lateral projections
from the presphenoid that surround the optic nerve and
fuse laterally, thereby creating the optic foramen. The
presphenoid, with its projecting orbitosphenoids, is homologous to the anterior portion of the sphenoidal body and
lesser wings in humans. The anterior portion of the presphenoid from joins the nasal septum, with the portion
between the pre- and postoptic roots commonly designated
as the interorbital septum. Crania from newborn and
adult mice were whole-mount-stained and examined for
the presence of these midline cranial base structures.
Whole-Mount Analysis
Newborn mice scored as 3H1 ⫹/⫹ showed a consistent
pattern of development in the bones of the midline cranial
base (Table 1, Fig. 3A). Ossification of the central bodies of
the basiocciptal, basisphenoid, and presphenoid was relatively complete, with only bridges of cartilage at the synchondroses remaining between each bone. The greater
wings were ossified in the lateral-most portions, but some
cartilaginous tissue remained between the central body of
the basisphenoid and ossified wing portions. The lesser
wings of the presphenoid showed some variation in the
amount of ossification: one mouse showed nearly complete
formation and fusion of the preoptic and postoptic roots of
the optic foramen, while others exhibited only the roots of
the wings from the central body of the presphenoid. At
minimum, all 3H1 ⫹/⫹ mice at this stage had a completely
ossified presphenoid body that was wide mediolaterally in
the middle and rostrally with a constricted area between
(Fig. 3A). The presphenoid in 3H1 ⫹/⫹ mice met anteriorly with the cribriform plate of the ethmoid, and the
orbital processes of the palatine bones were positioned
directly below the postoptic roots.
Mice scored as 3H1 Br/⫹ newborns showed different
developmental pattern from that of the ⫹/⫹ newborn mice
(Table 1, Fig. 3B). All 3H1 Br/⫹ mice at this age exhibited
a malformed presphenoid. The cartilage at the presphenoidal synchondrosis became greatly reduced mediolaterally towards the presphenoid body. A small central portion
of the body was ossified, however, the lateral portions of
the middle body, where it was wide in normal mice, was
totally lacking as bone or cartilage in 3H1 Br/⫹ mice. The
mediolateral restriction of this area during chondrogenesis seemed to have impaired the formation of the lateral
portions of the body. Furthermore, the normally wide rostral portion of the presphenoid body was also restricted
mediolaterally. The reduction in presphenoid body width
seemed to have an affect on normal orbitosphenoid development. Small isolated sphericals of bone rested on the
orbital processes of the palatines in Br/⫹ mice; these were
likely the postoptic roots of the optic foramina. The preoptic roots were not present in any of the Br/⫹ mice
examined with the exception of one individual showing
anterior rims completely formed in cartilage but with ossification still limited to the mid-body. The basisphenoid
body in 3H1 Br/⫹ mice was similar to the ⫹/⫹ mice except
for an anterior indentation in the posterior body at the
spheno-occipital synchondrosis. This seemed to be related
Fig. 3. Schematic of the interior cranial cavity of a newborn mouse
viewed from the superior perspective. A: The cranial base in 3H1 ⫹/⫹
mice shows a typical arrangement of bony and cartilaginous features,
with a well developed presphenoid and lesser wings (arrow). B: The 3H1
Br/⫹ mutant shows an ossifying postoptic root (arrow), but a severely
diminished presphenoid with no evidence of lesser wings. C: The pres-
phenoid and lesser wings are completely absent in the 3H1 Br/Br mutant
(*), while the nasal septum shows a large cleft (double arrow). Bo,
basioccipital; bs, basisphenoid; gw, greater wing; lw, lesser wing; n,
nasal septum; po, postoptic root; pr, preoptic root; ps, presphenoid;
pss, presphenoidal synchondrosis; sos, spheno-occipital synchondrosis. Bar ⫽ 0.5 mm.
to the occasional presence of an anterior midline split in
the synchondrosis directly adjacent to the indentation in
the basisphenoid body. The greater wings of the basisphenoid and basioccipital in newborn 3H1 Br/⫹ mice showed
morphology similar to 3H1 ⫹/⫹ mice.
Mice scored as 3H1 Br/Br mutants showed a normal
basioccipital; however, the basisphenoid was deficient anteriorly (Table 1, Fig. 3). The most obvious dysmorphology
was the complete absence of the presphenoid and presphenoidal synchondrosis (Fig. 3C). As a result, the orbitosphenoid and the pre- and postoptic roots were also absent
(Fig. 3C). Anterior to this region, the nasal capsule was
present; however, a nasal septum was absent and the
capsular cartilage was cleft throughout its length (Fig. 4).
All postnatal mice scored as 3H1 ⫹/⫹ exhibited cranial
base morphology consistent with normal morphology as
documented by Bateman (1954). Ossification of midline
cranial base structures was complete in all 3H1 ⫹/⫹ animals examined, with the exception of the petrosal processes of the basisphenoid in Day 17 or 18 individuals.
All postnatal 3H1 Br/⫹ mice exhibited a malformed
presphenoid that lacked lesser wings, with associated pre-
and postoptic roots (Table 1, Fig. 5). The presphenoid body
was restricted mediolaterally along its entire length and
decreased in length anteroposteriorly. Frequently the
body was positioned obliquely to the midsagittal line. A
small projection extending above the basisphenoid was
also occasionally observed (Fig. 5). The ethmoid often
fused with the anterior end of the presphenoid, and the
preoptic roots and interorbital septum were not present in
any of the individuals examined. The postoptic roots were
occasionally present, but only as isolated, malformed bony
projections that appeared attached to the sphenopalatine
processes and were not connected to the presphenoid body
(Fig. 5). The occurrence of a presphenoidal synchondrosis
was limited, but, when present, it was mediolaterally reduced and oval (rather than flat and rectangular) in
shape, and synostoses sometimes formed laterally between the presphenoid and basisphenoid (Fig. 5). The
basisphenoid body was reduced mediolaterally where it
met the presphenoidal synchondrosis, but widened to normal proportions posteriorly, similar to what was seen in
the newborn animals (Fig. 5). The posterior basisphenoid
body exhibited an anterior indentation, as observed in the
Basisphenoid - body
P/A, present/absent; N/A, normal/abnormal.
Presphenoid - preoptic
root of
Presphenoid - postoptic
root of
Presphenoid - body
Basisphenoid alisphenoids
Split in mid-line
Split in mid-line
Newborn Mice
Normal or absent
0/12 Isolated, ossified
sphericals not
attached to body
0/12 Reduced mediolaterally
in anterior
0/12 Reduced mediolaterally
Reduced in size;
deflected from midline
Mediolaterally reduced;
deflected from midline
Reduced in anterior
TABLE 1. Results of whole mount morphological observations
0/16 Reduced
0/12 Absent or isolated,
malformed bony
4/12 Disconnected from
presphenoid body
0/14 Absent or reduced
0/16 Projects above
basisphenoid or
reduced in size
and malformed
1/15 Buckled causing
inferior flexion
0/16 Reduced
2/14 Reduced in length
Adult Mice
newborn specimens. Frequently cartilage from the basioccipital synchondrosis filled the indentation, but it was also
occasionally observed to be vacant of bone or cartilage. All
3H1 Br/⫹ mice had retrognathic faces and the appearance
of globular vaults relative to the more dolichocranic ⫹/⫹
Microscopy demonstrated that the nasal capsule of newborn 3H1 Br/Br consisted of typical hyaline cartilage, as
indicated by type II collagen staining. A continuous perichondrium lined the cartilage, and the chondrocytes demonstrated a typical lacunar arrangement. A nasal septum
was absent, and the cranial base remained cleft throughout the length of the nasal capsule (Fig. 6). The newborn
3H1 Br/Br mice showed severe secondary palate clefting
while retaining a well formed median nasal prominence
(Fig. 7). The 3H1 Br/⫹ mice did not show anterior cranial
base or palatal clefting, but the sphenoethmoidal region
was diminished in size and shape, as previously reported
(Lozanoff et al., 1994). Ectopic cartilage formed in the area
of the crista galli and presphenoid of the heterozygotes
(Fig. 8).
Karyotype analysis revealed that the 3H1 Br/Br mice
did not have any gross aberrations, such as trisomies,
megabase deletions, or translocations (Fig. 9). Similarly,
G-banding failed to reveal any differences between 3H1
⫹/⫹ and Br/Br chromosomal arrangements (Fig. 9).
Microsatellite Mapping Analysis
Fig. 4. Whole-mount-stained preparations of the newborn nasal capsule (see schematic in Fig. 3 for reference). A: The 3H1 ⫹/⫹ mice display a
long nasal capsule with a well-defined nasal septum medially, as well as a
preoptic root (pr) and lesser wing (lw) extending laterally from the presphenoid (ps) and the presphenoidal synchondrosis (pss) is present caudally. B:
The 3H1 Br/⫹ mouse also shows a nasal septum (ns) medially, but a deeper
cleft between the anterior nasal cupulae is seen compared to the 3H1 ⫹/⫹
condition (arrowhead). Ectopic cartilage is present in the sphenoethmoidal
region (ec); a small postoptic root (po) is also present, but remains unconnected to the narrow presphenoid (ps). C: The Br/Br mutant shows a
complete midline cleft of the nasal capsule (nc), and completely lacks a
nasal septum (arrows) and presphenoid (*). Bar ⫽ 0.5 mm.
Microsatellite mapping established the order of loci as
D17Mit128 – D17Mit122 – D17Mit189 – D17Mit190, Br,
D17Mit155 – D17Mit221 – D17Mit123 (Table 2). Seven
recombinants were found in the 103 mice scored for
D17Mit128 and D17Mit122, one recombinant was found
in the 108 mice scored for D17mit122 and D17Mit189, one
recombinant was present in 104 mice scored for
D17Mit189 and D17Mit190, 0 recombinants were found in
107 mice scored for D17Mit190 and Br, 0 recombinants
occurred in 107 mice scored for Br and D17Mit155, and
three recombinants were found in 93 mice scored for
D17Mit155 and D17Mit221. Four recombinants were
found in the 100 mice tested for both D17Mit221 and
D17Mit123. Genetic distances (in cM) between the loci
were calculated as D17Mit128 – (6.79 ⫾ 2.48) –
D17Mit122 – (0.92 ⫾ 0.92) – D17Mit189 – (0.96⫾ 0.96) –
D17Mit190, Br, D17Mit155 – (3.23 ⫾ 1.83) – D17Mit221 –
(4.00 ⫾ 1.96) – D17Mit123. The order of the microsatellite
loci and their genetic separation were consistent with
those for mouse chromosome 17 (http://www.informatics., and all markers showed large and significant
LOD scores (Table 2; Fig. 10). On the basis of these data,
Br mapped to the interval of D17Mit189 and D17Mit221
representing a genetic distance of 4.19 ⫾ 2.79 cM.
Segregation analysis showed that offspring from 3H1
Br/⫹ reciprocal matings displayed one of three external
craniofacial morphologies (median midfacial cleft (Br/Br),
midfacial retrognathia (Br/⫹), and normal midfacial prognathism (⫹/⫹)). The mice with median facial clefts died
soon after birth, presumably because of their inability to
suckle as a result of the cleft. Previous reports also showed
that the 3H1 Br/Br external craniofacial morphology did
Fig. 5. Young adult (Day 17) cranial base synchondroses in (A) 3H1
⫹/⫹ and (B) Br/⫹ mice viewed from the ventral aspect (see schematic in
Fig. 3 for reference). A: The 3H1 ⫹/⫹ animal, when viewed from the
ventral aspect, and caudally to rostrally, shows basioccipital (bos), spheno-occipital (sos), and presphenoidal (pss) synchondroses, with the
nasal septum positioned rostrally and sagittally. B: The 3H1 Br/⫹ mutant
displays basiocciptal (bos) and spheno-occiptial (sos) synchondroses,
as well as a nasal septum (ns), but lacks a presphenoidal (pss) synchondrosis. The cranial base is viewed from the dorsal aspect in (C) 3H1 ⫹/⫹
and (D) Br/⫹ mice. C: The 3H1 ⫹/⫹ mice display a basiocciput (bo) and
basisphenoid (bs) with greater wing (gw) projections, while the developing presphenoid bone (ps) shows preoptic (pr) and postoptic (po) roots,
and lesser wings (lw) projecting laterally. The Br/⫹ young adult also
shows a basisphenoid (bs) with greater wings (gw), displaying a normal
appearance; however, the presphenoid (ps) is diminished in size and
extends ventrally to the basisphenoid (bs), lacking a synchondrosal
connection. Lesser wings (lw), and pre- (pr) and postoptic (po) roots are
completely absent (*). In fact, the optic nerve (on) was secondarily
stained and is seen to lack any bounding bony foramen. Bar ⫽ 0.5 mm.
not occur in reciprocal matings between 3H1 Br/⫹ and
3H1 ⫹/⫹ animals, nor was gender preferentially affected
(Ma and Lozanoff, 1993). Thus, Br showed a high degree of
penetrance and expression, while inheritance of this locus
was consistent with that of an autosomal semidominant
lethal trait.
The Br mutation arose in the 3H1 germ cell line as a
result of overexposure to gamma radiation, and it was
initially mapped to the distal portion of chromosome 17
(Searle, 1966; Beechey et al., 1997). The majority of radiation-induced mutations in mouse germ cells result in
gene deletions by causing double-stranded breaks in DNA
that the germ cell is unable to rejoin (reviewed by Abrahamson and Wolff, 1976; Sankaranarayanan, 1991, 1999).
However, the telomere may heal some breaks and confer
some degree of protection, particularly in the distal portions of a chromosome (Slijepcevic and Bryant, 1998).
Evidence suggests that the mammalian protein DNA polymerase micro (pol micro) forms discrete clusters following
radiation exposure that could stabilize the free ends (Mahajan et al., 2002). Even so, overexposure to gamma radi-
ation is likely to damage a chromosome, if not completely
delete a portion, since the vast majority of radiation-induced germ line mutations result in loss of function, and
demonstrate haploinsufficiency in the heterozygote and
lethality in the homozygous state (Sankaranarayanan,
1991, 1999). Although base substitutions, frameshifts,
and point deletions do occur, these types of mutations are
much less frequent as a result of radiation overexposure,
while insertions seldom occur (Grosovsky et al., 1988). The
radiation-induced nature of Br, as well as its inheritance
pattern and its position near the telomere of chromosome
17, was consistent with a repaired radiation-induced double-stranded break following a deletion.
The homozygous mutant consistently expressed specific
phenotypic features, such as the absence of major internal
midline features of the anterior cranial base (including the
nasal septum, presphenoid, and presphenoidal synchondrosis), while the basisphenoid was malformed rostrally.
Additionally, the primary and secondary palates did not
form in the 3H1 Br/Br mutant, reflecting another major
defect in the morphogenesis of the midline. Previously,
Fig. 6. Sequential sections through the nasal capsule of (A–C) 3H1
⫹/⫹ and (D–F) Br/Br newborn mice stained for type II collagen. The
normal animal shows a well formed nasal septum (n), while the mutant
displays a deep median facial cleft (arrow) separating the fully developed
lateral portions of the nasal capsule. More caudally, small bridges of
cartilage connect the two lateral components of (F) the nasal capsule,
but typically the two lateral halves maintain separate perichondria. Bar ⫽
0.1 mm.
Singh et al. (1998) showed that these palatal defects were
associated with structural hypoplasia, which indicated a
cessation of growth during the stage of murine primary
palate development (E10 –E12). All 3H1 Br/⫹ animals
displayed midfacial retrognathia externally, consistent
with previous reports (Ma and Lozanoff, 1993). Internally,
the presphenoid and nasal septum were both present, but
malformed and ectopic cartilage formation occurred in the
surrounding areas. The 3H1 ⫹/⫹ mice lacked these dysmorphic features. These observations are consistent with
a gene dosage effect of Br on craniofacial midline tissues,
since the heterozygote reflects haploinsufficiency, while
the homozygote mutant displays a more extreme phenotype consistent with a double allelic deletion.
Craniofacial syndromes that include median facial cleft
as a diagnostic feature have long been recognized as occurring in response to a dysplasia of the frontonasal prominence (Sedano et al., 1970). In humans, the clinical condition that includes median facial cleft, hypertelorism,
broad nasal root, and cranium bifidum is generally categorized as frontonasal malformation (FNM) (Sedano and
Gorlin, 1988). The 3H1 Br/Br mouse showed features consistent with frontonasal dysplasia, since the derivatives of
the frontonasal prominence were affected. However, the
absence of cranial base structures anterior to the basisphenoid in 3H1 Br/Br mutants suggests that the mutation may exert an effect on the prechordal mesoderm. In
the mouse, the prechordal plate forms as the head mesoderm and foregut endoderm fuse (Tam and Behringer,
1997). The prechordal plate interacts with the developing
neural tube that is undergoing rapid growth during neurulation. This process is regulated by many developmental
genes that affect craniofacial morphology. For example,
mice with null mutations for Gsc showed craniofacial defects in the vomer, palate, and sphenoid bones (RiveraPerez et al., 1995; Belo et al., 1998), while Gsc-1–/– mouse
embryos with hnf3␤ haploinsufficiency showed severe
ventralization of the brain along with reductions or loss in
Fig. 7. Light micrograph of newborn (A) 3H1 ⫹/⫹ and (B) Br/Br mice.
In the mutant mouse, the anterior cranial base region shows a severely
reduced lateral palatal shelf (p), which remains vertically oriented and
lateral to the tongue (t), resulting in cleft secondary palate (compared to
the normal condition, wherein a broad palate is fused horizontally). A well
formed medial nasal prominence (mn) is retained, while midline cranial
base cartilaginous tissue is completely absent in the mutant (compared
to the normal animal, in which a well formed posterior cartilaginous nasal
septum (ns) is present. Bar ⫽ 1.0 mm.
TABLE 2. Recombinant number (X), sample
size (N), map distance (Map), and LOD score with
associated probability (P) for the micro-satellite
mapping analysis
6.79 ⫾ 2.48
0.92 ⫾ 0.92
0.96 ⫾ 0.96
0.00 ⫾ 0.00
0.00 ⫾ 0.00
3.23 ⫾ 1.83
4.00 ⫾ 1.96
Fig. 8. A: Type II collagen staining of the mid-nasal septum region(s)
in a 3H1 ⫹/⫹ newborn mouse shows distinct perichondrial staining
(arrowheads). B: In contrast, the 3H1 Br/⫹ nasal septum shows ectopic
cartilage formation, and lacks a clearly defined perichondrium (arrows).
Bar ⫽ 0.5 mm.
Fig. 9. A comparison of representative karyotypes with G-banded (A)
3H1 ⫹/⫹ and (B) Br/Br chromosomes failed to reveal any gross aberrations.
expression of Sonic Hedgehog (shh) and fgf-8 (Filosa et al.,
1997). Similarly, shh is a major candidate involved in the
process midline formation, and failure of its expression
leads to abnormal patterning of the neural tube, resulting
in holoprosencephaly (HPE) and hypotelorism (Chiang et
al., 1996; Hammerschmidt et al., 1996; Muenke and
Beachy, 2000). Interestingly, Hu and Helms (1999) reported that decreased expression of shh in the anterior
midline tissues is associated with HPE and hypotelorism,
while shh overexpression is associated with widening of
the frontonasal prominence and hypertelorism. Since 3H1
Br/Br mutants showed median facial clefting characteristics and frontonasal dysplasia, the effect of Br could be
directed at the anterior midline tissue at the earliest
stages of craniofacial development, possibly through an
interaction with shh.
The mutant craniofacial morphology seen in the mutants resembles that reported for retinoic acid deficiency
(RAD) induced by pharmacological or genetic experimental techniques. A previous study (Marshall et al., 1996)
found that biologically active retinoids were present in
developing mouse embryos from gestational day 7.5, and
that they functioned to regulate neural crest migration,
frontonasal prominence development, and cellular differentiation. The activity of retinoids has been reported to be
mediated by RAR-RXR heterodimers, and RA-synthesiz-
Fig. 10. Schematic of the proposed location of Br based on the
microsatellite mapping analysis.
ing and catabolic enzymes localized in specific tissues
(McCaffery et al., 1996; Zhao et al., 1996; Niederreither et
al., 1997; Moss et al., 1998; ). Most RAR and RXR isoforms
are expressed differentially (Lohnes, 1999) and function
through a host of mechanisms, including apoptosis (Alles
and Sulik, 1990), repatterning (Kessel and Gruss, 1991),
altered differentiation (Agarwal and Sato, 1993), neural
crest cell migration (Lee et al., 1995), and increased proliferation (Leber and Denburg, 1997). RAD embryos exhibited foreshortened skulls, anophthalmia, cleft palate,
and cranial base dysmorphology (Wilson et al., 1953; Morriss-Kay and Sokolova, 1996; Mendelsohn and Baselga,
2000). Dickman et al. (1997) found that RA antagonism at
gestational day 8 resulted in frontonasal dysplasia, and
appeared to be related to alterations in neural crest fate
specification. Frontonasal dysplasia was reported to be
present in RAR␣/␥ null mutants, which also showed aplastic and ectopic cartilaginous elements in the rostral cranial base and midline (Lohnes et al., 1994) resembling the
ectopic cartilage displayed by 3H1 Br/⫹ heterozygotes.
Renal aplasia or hypoplasia (features common to 3H1
Br/⫹ and Br/Br mice (Ma and Lozanoff, 1993; Lozanoff et
al., 2001)) also occurred in these RAR null embryos (Men-
delsohn et al., 1994, 1999). Therefore, Br may be related to
abnormal endogenous RA regulation, with effects on neural crest morphogenesis. The recent observation that the
presphenoid and basisphenoid arise from neural crest exclusively in Wnt-Cre/R26R mice suggests that the defect
in Br may result from a neurocristopathy (McBratney and
Morris-Ray, personnal communication).
DeMyer (1967, 1975) was the first to recognize that
median facial cleft occurrence with hypotelorism frequently involves forebrain defects (as seen, for example, in
HPE), while median facial cleft incidence with hypertelorism (as seen in FNM) typically does not involve neural
malformations. Increasing evidence suggests that HPE
and FNM lie at opposite ends of a craniofacial morphogenetic continuum. Although little is known concerning the
genetic transmission of FNM, increasing evidence suggests that it results from overexpression of shh (Hu and
Helms, 1999) or RA deficiency (Lohnes et al., 1994). The
specific mechanism remains unclear; however, excess RA
induced RAR binding to RARE on the shh gene, thereby
down-regulating shh and downstream genes that cause
HPE. Conversely, RA deficiency resulted in shh up-regulation associated with frontonasal dysplasia (Franco et al.,
1999). It would appear that the gene responsible for the Br
phenotype may be involved in disturbing the balance between RA and shh, resulting in frontonasal dysplasia.
As a result of the microsatellite linkage analysis, Br was
placed on the distal end of chromosome 17 in the region
around D17Mit155. Physical mapping placed potentially
important genes regulating craniofacial development in
this region. Six3 (sinus oculus-related homeobox 3 homologue in Drosophila) was localized to 45.5 cM on mouse
chromosome 17, and it was homologous to human gene
SIX3 located on Chr 2 (p21–p16). This gene was reported
to be a candidate for HPE2, and is considered to be essential for development of the anterior neural plate and eye
(Wallis et al., 1999). Alk (anaplastic lymphoma kinase) is
located at cM position 50.0 on murine chromosome 17, and
is homologous to the human ALK gene located on Chr 2
(p23). ALK was reported to be a dosage-dependent gene
that is widely expressed in the neonatal brain, and presumably plays an important role in the development of the
neurulating brain (Iwahara et al., 1997). Zfp161 (zinc
finger protein 161) is located at 41.0 cM on murine chromosome 17, and is homologous to the human ZFP161 gene
located on Chr 18p11.21, near the transforming growth
interacting factor (TGIF) gene. The mouse tgif gene has
not been mapped; however, based on homology, the mouse
Zfp161 gene has been suggested to be analogous to TGIF,
which was also a candidate gene associated with HPE4
(Sobek-Klocke et al., 1997). TGIF was reported to be a
homedomain protein that may repress RA-regulated gene
transcription. Thus, the Br locus contains genes that,
when mutated, could be expected to lead to frontonasal
dysplasia and HPE. Current work is focused on the physical mapping of Br, as well as its relationship with RA
expression and processing.
All of the adult 3H1 Br/⫹ mice had facial retrognathia
and altered anterior vault morphology, but the cranial
base morphology was somewhat more variable, substantiating previous reports (Lozanoff, 1993). This finding suggested that the presphenoid defect was localized to the
cranial base early in development. During subsequent
development, integration between craniofacial elements
increased, causing systemic and coordinated changes in
the entire cranium, stemming from the defective cranial
base. Midfacial retrognathia was consistently present in
the heterozygous adults, reflecting the importance of the
presphenoid in determining normal prognathism and occlusion (Lozanoff et al., 1994; Lozanoff, 1999). Future
work will be directed toward determining patterns of
craniofacial growth integration as the result of a deficient
presphenoid in the 3H1 Br/⫹ mutant.
This study was supported in part by Medical Research
Council grant number 1069 and Hawaii Community
Foundation grant number 20012653 (to S.L.), and a Sigma
Xi travel grant (to B.M.). Jayne Johnston assisted with the
karyotype analysis, and Dailin Yee provided assistance
with a portion of the microstatellite data analysis. Beth K.
Lozanoff provided the schematic in Fig. 3. We thank Drs.
Daniel E. Lieberman and Charles Boyd for providing advice, and access to facilities necessary to complete this
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