The Muenke syndrome mutation (FgfR3P244R) causes cranial base shortening associated with growth plate dysfunction and premature perichondrial ossification in murine basicranial synchondroses


  • Jason Laurita,

    1. Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Eiki Koyama,

    1. Division of Orthopedic Research, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    2. Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Bianca Chin,

    1. Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    2. Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Jesse A. Taylor,

    1. Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    2. Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Gregory E. Lakin,

    1. Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Kurt D. Hankenson,

    1. Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Scott P. Bartlett,

    1. Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    2. Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
    Search for more papers by this author
  • Hyun-Duck Nah

    Corresponding author
    1. Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
    2. Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
    • 1116G/ARC, The Children's Hospital of Philadelphia, 3615 Civic Center Blvd., Philadelphia, PA 19104

    Search for more papers by this author


Muenke syndrome caused by the FGFR3P250R mutation is an autosomal dominant disorder mostly identified with coronal suture synostosis, but it also presents with other craniofacial phenotypes that include mild to moderate midface hypoplasia. The Muenke syndrome mutation is thought to dysregulate intramembranous ossification at the cranial suture without disturbing endochondral bone formation in the skull. We show in this study that knock-in mice harboring the mutation responsible for the Muenke syndrome (FgfR3P244R) display postnatal shortening of the cranial base along with synchondrosis growth plate dysfunction characterized by loss of resting, proliferating and hypertrophic chondrocyte zones and decreased Ihh expression. Furthermore, premature conversion of resting chondrocytes along the perichondrium into prehypertrophic chondrocytes leads to perichondrial bony bridge formation, effectively terminating the postnatal growth of the cranial base. Thus, we conclude that the Muenke syndrome mutation disturbs endochondral and perichondrial ossification in the cranial base, explaining the midface hypoplasia in patients. Developmental Dynamics 240:2584–2596, 2011. © 2011 Wiley Periodicals, Inc.


The P250R substitution mutation between the second and third extracellular Ig-like domains of fibroblast growth factor receptor 3 (FGFR3) causes Muenke syndrome, an autosomal dominant form of syndromic craniosynostosis (Moloney et al., 1997; Muenke et al., 1997). It is characterized by coronal suture synostosis and other craniofacial abnormalities, although its clinical presentation can be variable. Common but incompletely penetrant skull phenotypes include uni- and bi-lateral coronal synostosis, midface hypoplasia, downslanting palpebral fissures, ptosis of the eyelids, macrocephaly, dental malocclusion, and a highly arched palate (Muenke et al., 1997; Doherty et al., 2007).

FGFR3 is a high-affinity transmembrane receptor for many fibroblast growth factors (FGFs), and known to play a critical role in endochondral bone growth. FGFR3 exists as two major splice variants, IIIb and IIIc, with IIIc being the one predominantly expressed in proliferating chondrocytes (Deng et al., 1996; Ornitz et al., 1996; Rice et al., 2003). Studies of FgfR3 null mice showed that loss of FGFR3 activity was associated with increased rates of chondrocyte proliferation and lengthened chondrocyte columns in the epiphyseal growth plate of long bones (Colvin et al., 1996; Deng et al., 1996). Conversely, mice harboring a constitutively activating FgfR3 mutation showed decreased chondrocyte proliferation and differentiation associated with generalized skeletal hypoplasia (Naski et al., 1996, 1998). These studies concluded that FGFR3 is a negative regulator of endochondral bone growth. In line with the findings from animal studies, constitutively activating mutations of FGFR3 have been causally linked to deficient endochondral bone growth in several forms of dwarfism, including hypochondroplasia (N540L), achondroplasia (G380R), and thanatophoric dysplasia (R248C, K650E) in humans (Rousseau et al., 1994; Bellus et al., 1995a, b; Muenke and Schell, 1995). Earlier studies have suggested that constitutively active FGFR3 inhibits chondrocyte proliferation by dysregulating the Indian hedgehog (IHH)-parathyroid hormone related peptide (PTHrP) feedback signal (Chen et al., 2001), and STAT signaling pathways (Krejci et al., 2009). Sustained activation of the MAPK pathway, which is expected from constitutively activating mutant FGFR3, has also been shown to inhibit chondrocyte differentiation and arrest endochondral bone growth (Murakami et al., 2000; Zhang et al., 2006).

Muenke syndrome is unique among disorders linked to FGFR3 mutations in that long bone growth appears largely unaffected. Instead, it is primarily known for abnormal suture formation and defective limb patterning, such as broad thumbs, brachydactyly and clinodactyly, carpal and tarsal coalitions, thimble-like phalanges, and coned epiphyses (Reardon et al., 1997; Graham et al., 1998; Lajeunie et al., 1999; Sabatino et al., 2004). The clinical distinction between Muenke and other FGFR3-related syndromes may be explained by the unique molecular behavior of FGFR3 harboring the Muenke mutation. A study using surface plasmon resonance and X-ray crystallography showed that the P250R mutation significantly enhanced FGFR3IIIc's affinity for unnatural ligands, FGF2 and FGF9, as well as one of its cognate ligands, FGF1 (Ibrahimi et al., 2004). The cranial suture phenotype in Muenke syndrome is shared with Apert and Pfeiffer syndromes, which are caused by the P253R mutation of FGFR2 and the P250R mutation of FGFR1, respectively. The Pro to Arg substitutions also endow FGFR2IIIc and FGFR1IIIc with the ability to bind FGF9 with high affinity (Wilkie et al., 2001; Ibrahimi et al., 2004). Given that FGF9 is highly expressed in the cranial suture and increased FGF9 signaling causes craniosynostosis in mutant mice (Eks; Harada et al., 2009) and humans (Wu et al., 2009), FGF9 induced hyperactive/aberrant FGFR signaling in the cranial suture is likely the common pathophysiology that underlies all three craniosynostosis syndromes with Pro to Arg substitution in FGFRs.

Whereas suture pathobiology has been the primary focus of a majority of Muenke studies, another commonly described feature of the syndrome is maxillary hypoplasia with a depressed nasal bridge, a phenotype associated with deficient growth of the anterior cranial base (Muenke et al., 1997; Doherty et al., 2007). In contrast to the cranial vault that forms new bone primarily by intramembranous ossification, the cranial base grows in length by endochondral ossification of synchondroses where two opposing growth plate cartilages share a zone of resting chondrocytes. There are numerous synchondroses located in the cranial base with a wide range in closure times from birth to puberty. The intersphenoidal (ISS) and spheno-occipital synchondroses (SOS) are the primary postnatal growth centers for the cranial base. The ISS closes between 2 and 3 years of age and the SOS closes much later between 14 and 18 years (Madeline and Elster, 1995; Okamoto et al., 1996). Fusion of the SOS signifies the end of significant cranial base growth and midface development. In mice with a G374R substitution in FgfR3 (analogous to the human achondroplasia mutation), premature ossification of basicranial synchondroses has been observed (Matsushita et al., 2009).

To date, no study has looked into whether the Muenke mutation has any effect on the growth of basicranial synchondroses. In this study, using a knock-in mouse model for Muenke syndrome (FgfR3P244R), we demonstrate that the Muenke mutation leads to a decrease in the proliferating chondrocyte population, accelerated entry into the prehypertrophic stage coupled with reduced terminal differentiation of chondrocytes, premature perichondrial ossification of basicranial synchondroses, and reduced basicranial length. We also report that primary spongiosa bone formation was severely defective in the FgfR3P244R mouse cranial base, indicating disturbed transition from cartilage to bone during endochondral ossification.


Craniofacial Phenotypes Are Present Early in Juvenile FgfR3P244R Mice

Micro CT analysis of FgfR3P244R mice revealed that the cranial phenotype previously observed in adult mice (Twigg et al., 2009) was present in juvenile mice (Fig. 1). The characteristic loss of interdigitation and/or fusion of the premaxillary sutures and Class III malocclusion of the incisors can occur as early as postnatal week 1 (Fig. 1B,C,E,F). Snout deviation was observed in mutant mice at week 3 or later in the direction of the fused premaxillary suture (Fig. 1J). In cases of bilateral premaxillary fusion, snouts were either shortened without deviation or deviated in the direction of the more severely disrupted premaxillary suture (Fig. 1J,L). In all these cases, the premaxilla was visibly hypoplastic (Fig. 1E,K). Table 1 summarizes these craniofacial phenotypes in our sample set, a cohort of 66 FgfR3P244R mice at 1, 3, and 5 weeks of age, including 20 wild-type (WT), 20 heterozygous (FgfR3P244R+/−), and 26 homozygous (FgfR3P244R+/+) mice.

Figure 1.

Gross morphological changes in juvenile FgfR3P244R mutant mouse skulls. One- and 3-week-old homozygous FgfR3P244R mutant mice (FgfR3P244R+/+) (A–C,G–I) display shortening of the snout and malocclusion of incisors (green asterisk) (E,K), correlating with fusion of premaxillary sutures (yellow arrows) (F,L). These changes were observed only in some 1-week-old homozygous mutant, but relatively common in both homozygous and heterozygous (FgfR3P244R+/−) 3-week-old mutant mice. Skewing of the snout (red arrow) is visible in some 3-week-old mutant mice (J). Age matched wild-type mice (WT) did not show these phenotypes (A–C,G–I). The white arrow points patent premaxillary sutures.

Table 1. Summary of Craniofacial Phenotypes in FgfR3P244R Mutant Mice
Snout deviationFused PMMal-occlusion
1 WkWild type40/40/40/4
3 WksWild type80/80/81/8
5 WksWild type80/80/82/8

FgfR3P244R Mice Have Cranial Base Length Deficit Mostly Rostral to Basisphenoid

Craniomorphometric measurements were made for the full cohort of juvenile mice and adult mice (>8 weeks). Landmarks were identified on two-dimensional (2D) μCT sections, and distance measurements taken between them in 3D. The measurements for the total skull length (Fig. 2A) showed that homozygous FgfR3P244R skulls were significantly shorter than wild-type skulls from postnatal week 3 onward, and heterozygous skulls were significantly shorter than wild-type from week 5 onward (Data not shown). The skull length deficit in FgfR3P244R skulls was mostly anterior to the intersphenoidal synchondrosis (ISS) in juvenile stages, but it extends to basisphenoid later in adult stage (data not shown). The total skull length includes the measurements from structures other than cranial base. To determine if the growth of the basicranium itself was affected in FgfR3P244R skulls, we measured the basicranial length, the sum of presphenoid, basisphenoid, and basioccipital bone (Fig. 2B), as well as the lengths of the three individual basicranial bones (Fig. 2C). These measurements were compared between wild-type and mutant samples (Fig. 2C). Significant shortening of the basicranial length was observed from postnatal week 3 onward (Fig. 2B). Similarly, measurements for individual basicranial bones taken at week 3 showed significant shortening of the presphenoid (P < 0.01) and basisphenoid (P < 0.05) in homozygous mutants and subsequently also the presphenoid in heterozygous mutants at week 5 (Fig. 2C). There was no significant difference between genotypes in the length of the basioccipital bone (Fig. 2C). The reduced presphenoid length in homozygous mutants persisted into adulthood (P < 0.01), as does the shortening of the basisphenoid (P < 0.05) (Fig. 2C).

Figure 2.

Three-dimensional (3D) craniomorphometric analysis of the basicraium in FgfR3P244R mutant mice. Basicranial length measurements were made for all juvenile mice (n = 66) and adult mice (> 8 weeks; n = 12). A: Landmarks on the sagittal axis were identified as follows: 1, the most anterior point of the nasal bones; 2, the most anterior point of the presphenoid bone; 3, the anterior margin of the intersphenoidal (ISS); 4, anterior margin of spheno-occipital synchondroses (SOS); 5, the most anterior point of the foramen magnum (endobasion); 6, the most posterior point of the occipital bone (inion). Five measurements were made between these landmarks in each skull: rostral segment from landmark 1 to landmark 2, presphenoid (PS) from landmark 2 to landmark 3; basisphenoid (BS) from landmark 3 to landmark 4; basioccipital (BO) from landmark 4 to landmark 5, caudal segment from landmark 5 to landmark 6. (1-2)+(2-5)+(5-6) was considered the total skull length. B: Total basicranial length, the sum of PS, BS, and BO, was compared between genotypes at three time points (3, 5 and >8 weeks). C: To identify the source of cranial base shortening, each segment of the basicranium (PS, BS, and BO) was compared between genotypes at 3, 5, and >8 weeks. An asterisk (*) indicates a significant difference from wild-type (WT) with a P-value < .05.

Basicranial Synchondroses Prematurely Fuse in FgfR3P244R Mice

μCT of the SOS and ISS showed bony bridge formation in many of the FgfR3P244R mutants as early as postnatal week 3, but never in wild-type juvenile mice (Fig. 3). In wild-type mice, margins of the synchondrosis approached each other gradually and fused evenly across the entire length of the synchondrosis (Fig. 3A,D,G,J). Bridge formation in FgfR3P244R mutants was more prominent in the ISS than the SOS (Fig. 3). Nine of 10 of homozygous and 2/5 heterozygous mutants had formed bone across the ISS by week 3, where only 3/10 homozygous and 0/5 heterozygous mice had bridging across the SOS at this time point (Fig. 3E,F). By week 5, 12/12 homozygous and 4/8 heterozygous had ossification across the ISS, while 7/12 homozygotes and 1/8 heterozygotes had bony bridging across the SOS (Fig. 3H,I). In contrast, all wild-type ISS and SOS remained patent through week 5 (indicated with red and green asterisks in Fig. 3G). Both synchondroses were fully closed in mice older than 8 weeks irrespective of the genotype (Fig. 3J–L). The diagram illustrates locations of basicranial synchondroses and bones in the murine cranial base (M).

Figure 3.

Micro-CT images of a typical timeline of synchondrosis closure in wild-type and FgfR3P244R mutant mouse basicrania. A–L: Skull bases of wild-type (A,D,G,J), heterozygous mutant (B,E,H,K), and homozygous C,F,I,L) were imaged at four different time points: 1 week (A–C), 3 weeks (D–F), 5 weeks (G–I), and adult (>8 weeks, J–L). In all images, left is rostral and right is caudal. ISS is indicated with a red asterisk (open) or red arrowhead (bridged or closed) and the SOS with a green asterisk (open) or green arrowhead (bridged or closed). Scale bar = 500 μm. The diagram illustrates locations of and relationships among synchondroses and bones in the cranial base (M).

The degree of ossification or closure of basicranial synchondroses in our mouse sample set was assessed using a grading scheme devised by us as described above in the Experimental Procedures section and illustrated in Figure 4. Of note, synchondroses with the closure grade 3 or greater are unable to grow in the antero-posterior direction. The distribution of closure grades shown in Table 2 was plotted for the ISS and SOS of 1, 3, 5, and >8 (8–52) week-old mice of all three genotypes (Fig. 5). At 1 week of age, all mice presented Grade 1 (patent) synchondroses (excepting one homozygous FgfR3P244R mouse with a Grade 2 SOS), with margins clearly separated and no ossification centers visible. By 3 weeks of age, 10 of 11 homozygous mutant mice showed some level of ossification of the ISS (Grades 3, 4, and 5), whereas only 1 of 8 wild-type mice presented Grade 3 ISS. The mode value for closure grade for wild-type ISS was 1 at postnatal week 3, grade 2 at postnatal week 5, and grade 4 at >8 weeks (Fig. 5A,C,E). Meanwhile the mode ISS closure grade of homozygous mutants was higher in all age groups: grade 3 at 3 weeks, grade 4 at 5 weeks, and grade 5 at >8 weeks (Fig. 5A,C,E). The mode of SOS closure grade was not different between genotypes at 3 weeks (Grade 2) or at >8 weeks (Grade 4), although there was a larger variance for homozygous than wild-type mice (Fig. 5B,F). At 5 weeks of age, the SOS of all wild-type mice was still patent (grade 2), whereas the mode SOS closure grade for homozygous mice was 3 (Fig. 5D). These data revealed that longitudinal growth of the basicranium is ceasing or has ceased for most homozygous mice by postnatal week 5. Adult (>8 weeks) SOS showed that both wild-type and homozygous mutant mice attained mode closure grade of 4 (Fig. 5F). In heterozygous mutant mice, the ISS showed wide variability in degree of closure with the mode grade of 3 at 5 weeks and 5 at >8 weeks of age, whereas the SOS was quite similar to wild-type SOS in regards to closure timing and grades (Fig. 5B,D,F).

Figure 4.

A grading scheme for mouse basicranial synchondrosis closure. Three-dimensional (3D) micro-CT was used to develop the following grading scheme for mouse intersphenoidal (ISS; red arrow) and spheno-occipital synchondroses (SOS; green arrow): Grade 1, patent synchondrosis. Grade 2, patent but with at least one ossification center visible within the synchondrosis. Grade 3, margins of the synchondrosis are touching, or with a single area of slight ossification/bridging across the synchondrosis. Grade 4, significant ossification across the synchondrosis (>10% of the length of the synchondrosis fused), but synchondrosis not totally ossified. Grade 5, complete fusion with no apparent vestige remaining. The top and middle rows are superior views of the ISS and SOS, and the third row is the sagittal sectional view of the SOS. This scheme is based on the grading scheme developed for humans by others (Madeline and Elster, 1995). Scale bars = 500 μm.

Figure 5.

Graphic representation of the distribution of basicranial synchondrosis closure grades in wild-type and mutant mice. A–F: Graphs depict the percent of the sample with a given closure grade in 3-week (A,B), 5-week (C,D), and >8-week (E,F) old mice for intersphenoidal (ISS; A,C,E) and spheno-occipital synchondroses (SOS; B,D,F). For exact numbers of animals in each group, refer to Table 2. A clear trend for premature closure (grade >3) is shown as early as at week 3 for both homozygous and heterozygous mutant ISS (A) and week 5 for homozygous mutant SOS (D).

Table 2. Basicranial Synchondrosis Closure Grades in FgfR3P244R Mutant Mice
Intersphenoidal synchondrosis (ISS) closure grades
AgeGenotypesISS closure grade
1 weekWild type4/4----
3 weeksWild type6/81/81/8--
5 weeksWild type3/85/8---
Adult >8 weeksWild type--2/158/155/15
Spheno-occipital Synchondrosis (SOS) Closure Grades
AgeGenotypesClosure grade
1 weekWild type4/4----
3 weeksWild type1/87/8---
5 weeksWild type-8/8---
Adult >8 weeksWild type---10/155/15

Malocclusion Correlates With Premature Fusion of Specific Sutures/Synchondroses

A correlation between the type of malocclusion and the premature closure of certain sutures and synchondroses was observed as summarized in Table 3. Of 15 juvenile mice with patent premaxillary sutures but premature fusion of the ISS (defined as a closure grade 3 or higher at an age of 5 weeks or younger), 12 had Class III malocclusion of the molars only. On the other hand, 3 of 3 juvenile mice with patent ISS but prematurely fused/abnormal premaxillary sutures (defined as flattening/loss of interdigitation at an age of ≤ 5 weeks) presented incisor malocclusion only. Twelve mice with both premature ISS fusion and premaxillary fusion all had malocclusion of both the incisors and molars. Collectively, these data present the following correlations: (1) premature fusion of the ISS co-presents with molar malocclusion, and (2) premature fusion of the premaxillary sutures co-presents with incisor malocclusion.

Table 3. Correlation Between Specific Dental Malocclusions and Premature Fusion of the Pre-maxillary Suture (PM) and/or Intersphenoidal (ISS) in FgfR3P244R Mutant Mice
Fusion phenotypeNormal occlusion (n=34)Malocclusion
Molar only (n=17)Incisor only (n=3)Molar & incisor (n=12)
Premature ISS31200
Premature PM0030
Premature ISS+PM00012

Growth Plates of Basicranial Synchondrosis Are Dysfunctional in FgfR3P244R Mice: Decreased Chondrocyte Proliferation and Hypertrophy Are Associated With the Reduced Expression of Ihh

The ISS from 1- and 3-week-old mice were analyzed by histology and in situ hybridization to study the effect of the FgfR3P244R mutation at the cellular level. The wild-type ISS displayed characteristic growth plate zones composed of small, round, resting chondrocytes (rz), flattened proliferating chondrocytes (pz) and oval prehypertrophic chondrocytes (phz), and large mature hypertrophic chondrocytes (hz) (Fig. 6A,I). The ISS growth plate also showed spatially organized gene expression pattern that is characterized by the expression of FgfR3 in proliferating and early prehypertrophic chondrocytes, Type II collagen (Col II) in resting and proliferating chondrocytes, Type X collagen (Col X) in hypertrophic chondrocytes, and Matrix metalloproteinase 13 (MMP13) at the cartilage–osseous junction (Fig. 6A–D,I–L). At week 1, little difference was observed between FgfR3P244R mutant and wild-type ISS growth plate in terms of the zonal organization and chondrocyte marker gene expression profile (Fig. 6E–H). As the mice mature, both wild-type and mutant ISS became shorter in length with a reduced number of cells expressing Col II, Col X or MMP13 (Fig. 6I–L,M–P). The change was much more drastic in mutant (Fig. 6M–P) than wild-type (Fig. 6I–L). In 3-week-old mutant mice, the hypertrophic chondrocyte zone expressing Col X had nearly disappeared in the growth plate (Fig. 6O). In addition, the characteristic undulating appearance of ossifying cartilage septa at the cartilage–osseus junction shown in wild-type growth plate (Fig. 6I) was absent in the mutant (Fig. 6M).

Figure 6.

Disturbed endochondral ossification in FgfR3P244R intersphenoidal synchondrosis. A–T: Parasagittal serial sections of intersphenoidal (ISS) from 1-week-old (A–J) and 3-week-old (K–T) wild-type (A–E,K–O) and mutant (F–J, P–T) mice were processed for staining with fast green/Safranin O (A,E,I,M) and in situ hybridization with the indicated genes (B–E,G–J,L–O,Q–T). The stained sections from wild-type ISS show the presence of resting (rz), proliferative (pz), prehypertrophic (phz), and hypertrophic (hz) growth plate zones (A, I). Notice that the growth plate zonal structure is largely lost in 3-week-old mutant mouse ISS (P). In 1-week-old wild-type and mutant ISS, expression of FgfR3 is detected in the proliferating and early hypertrophic chondrocytes (B,G), Col II in resting and proliferating chondrocytes (C, H), and Col X in hypertrophic chondrocytes (D,I), and MMP13 in hypertrophic chondrocytes and the cells at the chondro–osseus junction (E,J). By postnatal week 3, FgfR3 expression was no longer detectable in both wild-type and mutant ISS (L,Q). Notice that, in the 3-week mutant synchondrosis (K–T), there is a significant reduction of Col II expressing proliferating chondrocytes (R) and Col X/MMP13 expressing hypertrophic chondrocytes (S,T).

In situ hybridization data from SOS revealed that the expression of Indian hedgehog (Ihh) was significantly lower in the mutant than wild-type already at week one (Fig. 7D,I; arrowhead) before visible changes in the growth plate structure (Fig. 7F) and Col II and Col X expression (Fig. 7G,H). As expected from reduced Ihh expression, Histone 4C (H4C), a marker for proliferating chondrocytes, was barely detectable in 1-week-old mutant SOS (Fig. 7J) compared with wild-type SOS (Fig. 7E). In 3-week-old mutant mice, similar to ISS abnormalities, the SOS growth plate showed significant reduction of Col II expressing resting/proliferating chondrocytes and Col X expressing hypertrophic chondrocytes (Fig. 7Q,R). In addition, the resting zone located in the middle of the synchondrosis became mostly occupied by the population of hypertrophying chondrocytes and was stained with fast green instead of safranin-O, revealing premature appearance of the secondary ossification center (Fig. 7P, yellow circle). The hypertrophic changes occurring in the resting zone of the mutant SOS was accompanied by the expression of Connective tissue growth factor (CTGF), a regulator of chondrocyte hypertrophy, and the drastic down-regulation of Sox9, a key molecule for the maintenance of chondrocyte immaturity (Fig. 7S,T). In contrast, the resting zone in the 3-week-old wild-type SOS had only a subtle change in the staining (Fig. 7K). Yet, the cells in this zone were still positive for Sox9 (Fig. 7O) and negative for CTGF (Fig. 7N).

Figure 7.

Abnormal endochondral ossification and accelerated secondary ossification center development in FgfR3P244R spheno-occipital synchondroses. A–T: Parasagittal serial sections of spheno-occipital synchondroses (SOS) from 1-week (A–J) and 3-week (K–T) wild-type (A–E,K–O) and FgfR3P244R mutant (F–J,P–T) mice were processed for staining with fast green/Safranin O (A,F,K,P) and in situ hybridization with the indicated genes (B–E,G–J,L–O,Q–T). Notice the specific expression of FgfR3 in the proliferating and the early hypertrophic chondrocytes in wild-type SOS (B), and marked reduction of Ihh expression in the prehypertrophic zone (I, arrowhead) and H4C expression in the proliferative zone (J, arrowhead) in mutant SOS. Note also that, in 3-week-old mutant SOS, the shortened growth plate is accompanied with the marked reduction of Col II and Col X -positive chondrocytes (Q,R), and the accelerated development of the secondary ossification center (P,S; indicated with a doted yellow circle) is recognizable by the activation of CTGF (S) and down-regulation of Sox-9 in the resting zone (T; indicated with a doted circle).

FgfR3P244R Mutation Accelerates Perichondrial Bone Formation in Basicranial Synchondroses

The chondrocyte zonal organization is also closely associated with bone formation along the perichondrium as shown in the 3-week-old wild-type ISS (Fig. 8A,B); resting and proliferating chondrocytes (Fig. 8B, yellow arrow) abutted on the perichondrial cell layer (Fig. 8B, red arrow), while the prehypertrophic and hypertrophic cells are flanked by the fast green-stained newly formed perichondral bone collar (Fig. 8A,B, yellow arrowheads). The perichondrial border flanking the proliferating and resting chondrocytes showed no appreciable bone formation (Fig. 8B, red arrow) and expression of early osteoblast marker genes, such as Type I collagen (Col I), Osteopontin (Op) and Osterix (Osx) (Fig. 8C–E, red arrows). By sharp contrast, fast green-stained bone had formed along the perichondrium of the FgfR3P244R mutant ISS (Fig. 8F,G), demonstrating that perichondrial bone formation is accelerated by the FgfR3P244R mutation. Resting and proliferating chondrocytes adjacent to the ossifying perichondrium were replaced by hypertrophying chondrocytes (Fig. 8G, yellow arrowhead). This excessive perichondrial bone formation corresponds to a precociously formed bony bridge across the 3-week-old mutant ISS initially detected by μCT (Fig. 3). As expected, mutant ISS showed increased and premature expression of genes involved in early osteogenic differentiation (Col I, Osteopontin, and Osterix; Fig. 8H–J). Not only was their expression increased but spanned the entire width of the synchondrosis in the area of the abnormal perichondrial bony bridge formation as opposed to being confined to the lateral edges of the synchondrosis as in the wild-type (Fig. 8C–E,H–J).

Figure 8.

Precocious perichondrial ossification of basicranial synchondrosis in FgfR3P244R mutant mice. A–J: Parasagittal serial sections of intersphenoidal (ISS) from 3-week-old wild-type (A–E) and mutant (F–J) mice were processed for staining with fast green/Safranin O (A,B,F,G) and in situ hybridization with the osteogenic genes such as Type I collagen (Col 1; C), Osteopontin (Op; D), and Osterix (Osx; C–E,H–J). Perichondrial regions of the wild-type and mutant ISS, which are indicated with boxes (A,F), are shown in a higher magnification (B,G). In wild-type, the membranous bone collar forms along the perichondrium flanking prehypertrophic and hypertrophic chondrocytes (B, yellow arrowheads). C–E: The perichondrium flanking resting chondrocytes (yellow arrow) remains unossified (red arrow) at this time, as indicated by the lack of osteogenic gene expression. In mutant ISS, perichondrial ossification is accelerated along the entire length of the synchondrosis cartilage indicated by the expression of osteogenic genes (H–J; yellow arrowhead). Notice that the chondrocytes flanking the perichondrium in mutant ISS have the appearance of prehypertrophic chondrocytes (G, yellow arrow).

Primary Spongiosa Formation Is Defective in FgfR3P244R Mutant Basicranial Synchondroses

The cartilage–osseous junction consists of hypertrophic chondrocytes, safranin-O staining positive cartilage matrix, fast-green staining positive bone matrix, osteoblasts and marrow cells (Fig. 9A,B). The cartilage–osseous junction in 1-week-old mutant SOS appeared largely normal (Fig. 9G), consisting of the primary spongiosa adjacent to the hypertrophic zone with an undulating surface intermixed with marrow (Fig. 9A). Its deficiency became apparent during the next 2 weeks; hypertrophic chondrocyte zone and the primary spongiosa were virtually absent in 3-week-old mutant SOS (Fig. 9H). The transition from cartilage to marrow was abrupt in mutant, creating a flat surface. The histological changes are reflected by significantly lower expression of bone markers and regulators, such as Col I, MMP9, MMP13, and Osterix, at the cartilage–osseous junction of the mutant SOS growth plate (Fig. 9C–F,I–L).

Figure 9.

Defective primary spongiosa formation in FgfR3P244R synchondroses. A–L: Parasagittal serial sections of spheno-occipital synchondroses from 1-week (A,G)- and 3-week (B–F,H–L) wild-type (A–F) and FgfR3P244R mutant (G–L) mice were processed for staining with fast green/Safranin O (A,B,G,H) and in situ hybridization with the indicated genes (C–F,I–L). Notice the significant reduction of hypertrophic chondrocyte zone (hz) and primary spongiosa (ps) at the chondro–osseous border in 3-week-old mutant SOS (H) compared with wild-type SOS (B), which is identifiable by the significant reduction of osteogenic marker genes (Col 1, Osx2) (I, L) and the genes required for chondro–osseous transition (MMP9, MMP13; J,K).


The present study showed that the mice harboring the mutation responsible for the Muenke syndrome (FgfR3P244R) presented with postnatal shortening of the cranial base, which was the result of a dysfunctional growth plate and premature perichondrial ossification of basicranial synchondroses. Mutant ISS and SOS displayed reduced numbers of proliferating chondrocytes, decreased expression of Ihh in prehypertrophic chondrocytes, absence of the hypertrophic zone in their growth plates, and the defective formation of the primary spongiosa. In addition, SOS displayed premature appearance of the secondary ossification center populated with CTGF expressing prehypertrophic chondrocytes. We propose that the Muenke mutation suppresses the IHH-PTHrP feedback signal, thereby promoting the entry of resting and proliferating chondrocytes into the prehypertrophic stage while reducing the population of proliferating chondrocytes in the synchondrosis. At the same time, the mutation inhibited prehypertrophic chondrocytes from undergoing terminal differentiation into Col X-expressing hypertrophic chondrocytes. It is likely that inability to maintain the hypertrophic zone is responsible for the defective primary spongiosa formation at the cartilage–osseous junction in the mutant cranial base. In the mutant synchondroses, especially ISS, precocious ossification of the perichondrium in the early postnatal stage led to the formation of a bony bridge across synchondrosis cartilage, bringing the growth of the anterior cranial base to an end.

Approximately two-thirds of the patients with Muenke syndrome reportedly have mild to moderate mid-face hypoplasia (Doherty et al., 2007), which is generally thought to be the result of the prematurely ossified coronal sutures. Indeed, unilateral coronal suture immobilization in an experimental animal leads to decreased bone growth at the coronal suture and shortening of the anterior cranial base with orbital asymmetry (Persing et al., 1986). However, the mid-face deficiency in Muenke syndrome cannot be explained solely by the presence of coronal synostosis. Comparison of the craniofacial phenotype between Muenke patients and non-Muenke patients with unilateral coronal synostosis showed that the severity of the craniofacial asymmetry was significantly greater in the Muenke group than the non-Muenke group, especially the anterior part of the skull and facial skeleton (Keller et al., 2007). This suggested that the Muenke mutation might have a direct effect on cranial base growth. Major postnatal lengthening of the cranial base takes place at the synchondroses that grow by endochondral ossification. The longitudinal growth of endochondral bones including synchondroses is profoundly affected by constitutively activating mutations of FGFR3. In human and mouse neonates harboring these mutations, basicranial synchondroses are completely closed (Deng et al., 1996; Matsushita et al., 2009). On the other hand, the Muenke syndrome mutation has been thought to disrupt skeletal growth by dysregulating the intramembranous bone formation at the suture. Our study demonstrates that the FgfR3P244R mutation also disrupts endochondral ossification in the postnatal cranial base, which is characterized by reduced chondrocyte proliferation, accelerated prehypertrophic changes and defective chondro–osseus transition. These changes are consistent with the phenotype expected from reduced IHH signals (Minina et al., 2002; Mak et al., 2008). Indeed, significantly reduced Ihh expression precedes detectable changes in the width and zonal arrangement of the growth plate in the mutant synchondrosis. In addition to the IHH-dependent indirect effect, the FgfR3P244R mutation may directly influence growth plate chondrocyte biology. It has been shown that FGF-FGFR3 signaling inhibits chondrocyte proliferation and differentiation through STAT1 and MAPK pathways (Sahni et al., 1999; Murakami et al., 2000, 2004; Legeai-Mallet et al., 2004; de Frutos et al., 2007). Thus, the FGFR3P244R mutant receptor may directly exert negative effects on endochondral ossification by means of overactive downstream signaling pathways. These two mechanisms are not mutually exclusive and may work in parallel.

The FgfR3P244R mutation disrupts and shortens the growth plate in both ISS and SOS. Interestingly, these growth plate changes in the synchondroses appeared to have a minor effect on cranial base growth. Instead, the deficiency of the cranial base length in the mutant mice was mostly limited to the anterior segment in association with the premature perichondrial bony bridge formation in the ISS. During normal development of the growth plate, perichondrial ossification is closely coordinated with endochondral ossification of the growth plate cartilage. Accordingly, we observed that bony margins of wild-type synchondroses advance evenly along the entire length of the synchondrosis through both endochondral and perichondrial ossification (Fig. 3). Despite that the periosteal bony collar formation progresses slightly ahead of the endochondral ossification of the growth plate, a prematurely forming perichondrial bony bridge is not seen in wild-type synchondroses (Figs 3, 8). In contrast, the perichondrium of mutant synchondroses ossifies long before growth plate cartilage is ossified, effectively terminating the lengthwise growth of the involved cranial base bones (Figs. 2, 3, 8). Endochondral ossification at the mutant chondro–osseus junction is clearly disturbed with drastically reduced terminally differentiated mature hypertrophic chondrocytes and primary spongiosa bone formation (Fig. 9). Therefore, it is unlikely that accelerated endochondral ossification is the primary cause for the aberrant bony bridge formation in mutant synchondroses.

Perichondrial ossification is coupled with prehypertrophic changes in the growth plate. The perichondrium remains unossified until the abutted resting chondrocytes begin to undergo hypertrophic changes. It is thought that IHH signals emanating from prehypertrophic chondrocytes play a key role in promoting differentiation of the osteochondro-progenitor cells located within the perichondrial region into the osteogenic lineage while suppressing the alternative chondrogenic pathway (Long et al., 2004; Young et al., 2006). However, prehypertrophic chondrocytes of the FgfR3P244R mutant synchondrosis showed drastically reduced expression of IHH. This suggests that factors/signals other than IHH must be responsible for the perichondrial bony bridge formation in the FgfR3P244R ISS. Activation of Erk-1/2, a downstream mediator of FGF/FGFR3 signaling, stimulates osteoblast differentiation and ossification of the perichondrium (Matsushita et al., 2009). Thus, FGFR3-mediated signaling may directly promote osteogenic differentiation of progenitor cells in the perichondrial region. Alternatively, FGF/FGFR3 signals may induce expression of osteogenic factors in perichondrial cells, stimulating perichondrial ossification. This suggestion is supported by earlier studies showing FGF/FGFR induction of BMP/TGFβ gene expression in osteogenic cells (Fakhry et al., 2005), and essential roles of BMP/TGFβ signaling in osteoblast differentiation in the perichondrium (Minina et al., 2002; Matsunobu et al., 2009).

The mouse model is interesting clinically, as the cranial base shortening in the mouse model closely mimics the human phenotype. Cephalometric analyses of Muenke syndrome patients show significant anterior cranial base shortening of an average of 8.4 mm (Ridgway et al., 2011). As in the mouse model, premature fusion of basicranial synchondroses likely contributes to the anterior cranial base deficiency in patients. However, due to deficient human CT data, this is difficult to verify. Interestingly, although midface hypoplasia is reported in a majority of Muenke syndrome cases, less than 25% of the patients require midface advancement surgeries (Honnebier et al., 2008). This may be explained by the fact that Muenke syndrome patients are heterozygotes. In the mouse model, the phenotype was clearly dependent on the dose of the mutant gene. Also, mild midface hypoplasia can be dentally compensated, not requiring surgical correction. Another explanation is retardation in mandibular antero-posterior growth that essentially camouflages a mild midface hypoplasia. In fact, cephalometric measurements showed that Muenke patients had shorter mandibular body length than normal individuals by an average of 12.9 mm (Ridgway et al.). We are currently studying the effect of FgfR3P244R mutation on the growth of the mandible and temporomandibular joint. In conclusion, this study reports for the first time that Muenke syndrome mutation negatively affects cranial base growth as a result of growth plate dysfunction and accelerated perichondrial ossification in the basicranial synchondrosis.



FgfR3P244R 129S6 mice, containing a knock-in mutation (c731g) in exon 7 of the FgfR3 gene, were kindly gifted by Dr. Twigg (Oxford University, Oxford, UK) (Twigg et al., 2009). For this study, 66 juvenile mice (1, 3, and 5 weeks old) and 57 adult mice (8–52 weeks old) were analyzed. For genotyping, polymerase chain reaction (PCR) was performed on the tail DNA with forward primer (5′-CTG TAC TCA AGG TAG GCT CT-3′) and reverse primer (5′-AGG GTA CTA ACT CAG CAG TC-3′). The PCR amplicon from the WT FgfR3 allele is expected to be approximately 500 bp, and that from the mutant allele, approximately 700 bp. PCR consisted of 35 cycles of 95°C for 45 sec, 55°C for 45 sec and 72°C for 45 sec, followed by 1 cycle of 72°C for 5 min. Mice were excluded from the study if genotype could not be reliably determined. All animal protocols for this study were approved by the Institutional Animal Care and Use Committee of the Joseph Stokes, Jr., Research Institute at the Children's Hospital of Philadelphia, in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Microcomputed Tomography (μCT)

Mouse skulls were debrided of soft tissue and fixed in 10% neutral-buffered formalin for a minimum of 24 hr before μCT analysis. They were then mounted in a foam scaffold in 15 or 50 ml of polyprophylene conical tubes containing 10% formalin. All samples were scanned at high-resolution with an isotopic voxel size of 12.5 μm, using a vivaCT 40 MicroCT scanner (Scanco Medical AG, Basseldorf, Switzerland), and analyzed using μCT v6.0 vivaCT software. Each skull consisted of between 900 and 1,200 12.5μm slices. The raw data from the μCT scans were compiled into 2D grayscale images that were then contoured to define the skulls. Binary images were generated using a threshold of 280. Virtual 3D models were then constructed and analyzed for morphological abnormalities and morphometric 3D measurements.

Gross Morphology

Snout deviation was defined as a significant curvature of the sagittal/frontal suture away from the midline when viewed from the top or front of the skull. Class III dental malocclusion was divided into incisor malocclusion and molar malocclusion. Incisor malocclusion was indicated by the mandibular incisors overlapping anterior to the maxillary incisors. The mandibular and maxillary first molars have three cusps, and the occlusion of these cusps determined molar malocclusion. We classified molar malocclusion as occlusion of the distal cusp of the first mandibular molar anterior to the mesial cusp of the first maxillary molar when viewed from the lateral aspect.

Synchondrosis Closure Grading

To objectively assess the degree of closure of the two major cranial base synchondroses, the ISS and SOS, a grading scheme was created using the following criteria (adapted from human synchondrosis grading scheme developed by Madeline et al. (Madeline and Elster, 1995): Grade 1, the margins of the synchondrosis are clearly separated with uninterrupted cartilage in between; Grade 2, the margins of the synchondrosis are separate with points of ossification visible within the synchondrosis; Grade 3, margins of the synchondrosis are <100 μm apart or touching, or with a single area of ossification/bridging across the synchondrosis (<10% of the length of the synchondrosis); Grade 4, significant areas of ossification across the synchondrosis (>10% of the length of the synchondrosis fused), but synchondrosis not completely closed, with obvious vestiges of the synchondrosis remaining; Grade 5, complete fusion with no apparent vestige remaining (Fig. 4).


The 3D linear measurements between landmarks within the mouse skulls were taken using the Distance 3D tool in the vivaCT software. Landmarks on the sagittal axis were identified on 2D slices as follows (Fig. 2): 1, the most anterior point of the nasal bones; 2, the most anterior point of the presphenoidal bone; 3, the anterior margin of the ISS; 4, anterior margin of SOS on the sagittal axis; 5, the most anterior point of the foramen magnum (endobasion); 6, the most posterior point of the occipital bone (inion).

Histology and In Situ Hybridization

Skulls were fixed in 4% paraformaldehyde, rinsed with DEPC (diethyl pyrocarbonate)-treated water, decalcified using 0.1 M Tris, pH 7.5 buffer containing 0.1% DPEC, 10% EDTA-4Na, and 7.5% polyvinyl pyrolidione (PVP) and processed for paraffin embedding. For routine histological analysis, 5μm sections were stained with Fast green/Safranin O using standard procedures.

For in situ hybridization, serial paraffin sections were pretreated with 10 μg/ml proteinase K (Sigma) for 10 min at room temperature, postfixed in 4% paraformaldehyde, washed in PBS containing 2 mg/ml glycine and treated with 0.25% acetic anhydride in triethanolamine buffer (Koyama et al., 2007). Sections were hybridized with antisense or sense 35S-labeled probes (approximately 1 × 106 disintegrations per second (DPM)/section) at 50°C for 16 hr. Mouse cDNA clones included: collagen I (Col 1; nt. 233-634; NM_007742); collagen II (Col2; nt. 1095-1344; X57982); collagen X (Col10a1; nt. 1302-1816; NM009925); osteopontin (Spp1; nt.1-267; AF515708); osterix (Sp7; nt. 40-1727; NM_130458); Ihh (nt. 897-1954; MN_010544; MMP 9 (NM_013599); MMP 13 (nt. 11-744; NM_008607); histone H4C (nt. 549-799; AY158963); connective tissue growth factor (CTGF) (nt. 680-840; NM_010217); and Sox 9 (nt. 116-856; NM_011448). After hybridization, slides were washed with 2× sodium chloride-sodium citrate buffer (0.3 M NaCl, 0.03 M Na-citrate, pH 7.0 SSC) containing 50% formamide at 50°C, treated with 20 μg/ml RNase A for 30 min at 37°C and washed three times with 0.1× SSC at 50°C for 10 min/wash. Sections were dehydrated with 70, 90, and 100% ethanol for 5 min/step, coated with Kodak NTB-3 emulsion diluted 1:1 with water, and exposed for 10–14 days. Slides were developed with Kodak D-19 at 20°C and stained with hematoxylin. Dark- and brightfield images were captured using a digital camera.

Statistical Analysis

For the statistical comparisons of craniomorphometric measurements between the three groups (homozygous, heterozygous, and wild-type), one-way analysis of variance (ANOVA) with a subsequent Bonferroni post hoc test was performed. In the graphs (Fig. 2), asterisks (*) indicate that the mean value of the heterozygous or homozygous group was significantly different from the wild-type group. A P-value of < 0.05 was considered significant. All data are presented as means ± standard deviation (SD).


We thank Drs. Steven R. Twigg and Andrew O. Wilkie (Oxford University, Cambridge, UK) for kindly providing FgfR3P244R mice and Dr. Maxmillian Muenke (National Human genome Research Institute, Bethesda, MD) for helpful suggestions. H.-D.N. was funded by NIH/NIAMS, and S.P.B. was funded by the Mary Downs Endowment Fund.