THE ANATOMICAL RECORD PART A 271A:291–302 (2003) Frontonasal Dysplasia in 3H1 Br/Br Mice BRANDEIS M. MCBRATNEY,1 EDITH MARGARYAN,2 WENBIN MA,3 ZSOLT URBAN,4 AND SCOTT LOZANOFF5* 1 Department of Anthropology, Harvard University, Cambridge, Massachusetts 2 Graduate Program in Cell and Molecular Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 3 Laboratory of Immunology, National Cancer Institute, Frederick, Maryland 4 Pacific Biotechnology Research Center, Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 5 Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii ABSTRACT 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 © 2003 WILEY-LISS, INC. 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. E-mail: email@example.com Received 30 May 2002; Accepted 11 November 2002 DOI 10.1002/ar.a.10034 Published online 7 March 2003 in Wiley InterScience (www.interscience.wiley.com). 292 MCBRATNEY ET AL. 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. MATERIALS AND METHODS 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. Animals 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 abnormal. Segregation of Br To test the inheritance pattern of Br, reciprocal matings between inbred parents (F7-10 generations) were under- Microscopy 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 FRONTONASAL DYSPLASIA IN 3H1 Br/Br MICE 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 293 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. jax.org. 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. med.buffalo.edu/mapmgr.html). 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. RESULTS 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 294 MCBRATNEY ET AL. 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 FRONTONASAL DYSPLASIA IN 3H1 Br/Br MICE 295 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 5/0 5/0 5/0 5/0 Spheno-occipital synchondrosis Basisphenoid - body 5/0 5/0 5/0 5/0 5/0 Palate P/A 12/0 12/0 12/0 1/11 12/0 12/0 12/0 12/0 12/0 12/0 P/A, present/absent; N/A, normal/abnormal. 5/0 5/0 5/0 5/0 5/0 Presphenoid - preoptic root of orbitosphenoid Presphenoid - postoptic root of orbitosphenoid Palatines 5/0 5/0 Presphenoid synchondrosis Presphenoid - body 5/0 5/0 Basisphenoid alisphenoids 5/0 5/0 Basioccipital N/A P/A Morphology ⫹/⫹ N/A Split in mid-line anteriorly Split in mid-line posteriorly Reduced anteroposteriorly Normal Description Newborn Mice Normal or absent 12/0 Normal 0/12 Isolated, ossified sphericals not attached to body 12/0 Normal 1/0 0/12 Reduced mediolaterally in anterior 0/12 Reduced mediolaterally 10/2 5/7 8/4 12/0 Br/⫹ 0/8 8/0 0/8 0/8 0/8 0/8 8/0 8/0 8/0 8/0 P/A – 0/8 – – – – 0/8 0/8 8/0 8/0 N/A Reduced in size; deflected from midline Absent Absent Absent Absent Mediolaterally reduced; deflected from midline Absent Reduced in anterior Normal Normal Description Br/Br 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 17/0 N/A ⫹/⫹ P/A TABLE 1. Results of whole mount morphological observations P/A 16/0 16/0 12/4 0/16 16/0 14/2 16/0 16/0 16/0 16/0 Description 0/16 Reduced anteroposteriorly 0/12 Absent or isolated, malformed bony projections 4/12 Disconnected from presphenoid body 0/14 Absent or reduced mediolaterally 0/16 Projects above basisphenoid or reduced in size and malformed – Absent Reduced anteroposteriorly 1/15 Buckled causing inferior flexion 0/16 Reduced anteroposteriorly 2/14 Reduced in length anteroposteriorly N/A 15/1 Br/⫹ Adult Mice 296 MCBRATNEY ET AL. FRONTONASAL DYSPLASIA IN 3H1 Br/Br MICE 297 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 ⫹/⫹ mice. Microscopy 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 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. jax.org), 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. DISCUSSION 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 298 MCBRATNEY ET AL. 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, FRONTONASAL DYSPLASIA IN 3H1 Br/Br MICE 299 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. 300 MCBRATNEY ET AL. TABLE 2. Recombinant number (X), sample size (N), map distance (Map), and LOD score with associated probability (P) for the micro-satellite mapping analysis Marker X N Map LOD P⬍ D17Mit128 D17Mit122 D17Mit189 D17Mit190 Br D17Mit155 D17Mit221 D17Mit123 7 1 1 0 103 108 104 107 6.79 ⫾ 2.48 0.92 ⫾ 0.92 0.96 ⫾ 0.96 0.00 ⫾ 0.00 19.9 30.0 28.9 32.2 .014 .005 .005 .001 0 3 4 107 93 100 0.00 ⫾ 0.00 3.23 ⫾ 1.83 4.00 ⫾ 1.96 32.2 22.2 22.8 .001 .009 .010 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- FRONTONASAL DYSPLASIA IN 3H1 Br/Br MICE 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 301 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. ACKNOWLEDGMENTS 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 study. LITERATURE CITED Abrahamson S, Wolff S. 1976. 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