Disruption of Axin2 and Fgfr1 in mice results in ectopic chondrogenesis in the sagittal suture
In humans and mice, the skull vault has seven sutures, and the patency (ability to remain open and continue to form bone) of these sutures is essential for proper growth regulation of the skull, especially during early postnatal stages (3
). Two of these are the sagittal (SAG) suture and the posterior frontal (PF) suture. The PF suture closes during a mouse’s lifetime, whereas the SAG suture and all other cranial sutures remain patent and bone continues to form at these sites as the animals grow (25
). In humans and mice, the PF suture is structurally unique and its development requires a specific mechanism (26
). This suture, residing between the frontal bones, is form a single mesenchymal interface, from which new bone forms, between two adjacent skull bones. The fusion of the PF suture’s endocranial layer may involve a mechanism different from that occurring at other sutures, because chondrocytes transiently appear only at the PF suture during the second week of postnatal development (28
). Like all other mouse cranial sutures, the ectocranial layer remains patent throughout life.
Because both WNT signaling and FGF signaling have been implicated in skeletal development and synostosis, we analyzed how these pathways interact during suture development and closure by monitoring skull development in postnatal mice deficient for Axin2 (Ax2−/−), in mice heterozygous for Fgfr1 (Fgfr1+/−), or in double-mutant mice (Ax2−/−; Fgfr1+/−) ().
Fig. 1 Ectopic chondrogenesis caused by loss of Axin2 and decreased FGFR1 induces ectopic chondrogenesis. (A to D, A′ to D′) Skeletal staining of the P7 skulls with AB shows that ectopic chondrogenesis (arrow) occurs in the SAG suture of Ax2 (more ...)
Alcian blue (AB) staining of skulls of postnatal day 7 (P7) mice revealed that the double-mutant SAG sutures displayed obvious morphological differences compared to those of the wild type, Ax2−/−
, and Fgfr1+/−
(). At P7, we detected a small AB-positive region indicative of ectopic deposition of connective tissue in the SAG sutures of 37% of Ax2−/−
skulls (), and all of the SAG sutures exhibited a larger area of ectopic chondrogenesis in Ax2−/−
animals examined (). Neither the skulls of the wild-type () nor Fgfr1+/−
() animals had detectable chondrogenesis. To confirm the presence of chondrocytes, in addition to staining with AB, we stained for chondrocyte markers SOX9 and type II collagen (25
). AB staining () and immunostaining of Sox9 () and type II collagen (COL II, ) in cranial sections confirmed that Ax2−/−
mouse skulls had ectopic chondrogenesis in the developing SAG suture. Additionally, this analysis revealed that the ectopic chondrogenic layer developed beneath the SAG suture, resembling the endocranial layer of the developing PF suture.
SAG sutures of mice with disrupted Axin2 and FGFR1 exhibit PF-like activity of the FGF and TGF-β pathways
The PF and SAG sutures exhibit differential activity of FGF2 and transforming growth factor–β (TGF-β) signaling, with high activity of these two pathways in the PF suture and limited activity in the SAG suture (29
). Therefore, we examined whether the PF-like SAG suture of the Ax2−/−
mice exhibited the FGF2 and TGF-β profile resembling that of the PF suture by analyzing the abundance of FGF2 and phosphorylated SMAD2 (pSMAD2, a marker of TGF-β activity) (). Analysis of P9 wild-type mouse skulls revealed FGF2 and pSMAD2 in the intramembranous ossification regions, including the osteogenic fronts and the area surrounding the bone plates, of both the PF and the SAG sutures (, black arrows). Additionally, FGF2 and TGF-β were abundant in the endocranial layer of the PF suture mesenchyme that was undergoing chondrogenesis (, red arrowheads). The number of FGF2- and pSMAD2-positive cells was higher in the PF suture compared to that in the SAG suture (). Furthermore, these chondrogenic increases in FGF2 and TGF-β signaling activities coincided with the chondrocyte-mediated abnormalities within the Ax2−/−
SAG sutures (, red arrowheads), suggesting their alterations into PF-like sutures in the double mutants. The numbers of FGF2- and pSMAD2-positive cells in the mutant SAG sutures were comparable to the numbers in the wild-type PF sutures ().
Fig. 2 Increased abundance of FGF2 and phosphorylated Smad2 coincides with development of the normal PF suture and the mutant SAG suture that exhibits a PF suture–like structure. Sutures of P9 WT and double-mutant mice were immunostained for FGF2 (A (more ...)
Alteration of WNT and FGF signaling results in craniosynostosis in mice
To determine whether disruption of Axin2 and Fgfr1 induces suture deformities resulting in craniosynostosis, we examined the SAG and PF sutures and skulls of mice at P50, a time when the PF sutures are normally closed. In the wild-type, Fgfr1+/−, and Ax2−/− mice, the SAG sutures were open at P50 (), whereas in the Ax2−/−; Fgfr1+/− mice, the SAG sutures were fused (). Similar to the PF suture at this stage in the wild-type animals (), the SAG suture also consisted of an ectocranial and an endocranial layer in the Ax2−/−; Fgfr1+/− (). The SAG suture was a single layer in wild-type, Fgfr1+/−, and Ax2−/− mice (). Furthermore, the synostosis occurred in the endocranial layer of the SAG suture of the double-mutant mice. Thus, whereas individual disruption of Axin2 or Fgfr1 does not alter the SAG suture, combined deficiency converts the SAG suture to one resembling the PF suture, leading to craniosynostosis.
Fig. 3 Craniosynostosis occurs in the SAG suture of Ax2−/−; Fgfr1+/− at P50. (A to D) Ventral view of the Alizarin red–stained skulls reveals the closure of the SAG suture (arrowhead) in the Ax2−/−; Fgfr1+/− (more ...)
Ectopic endochondral ossification is the likely cause of synostosis due to altered Axin2 and Fgfr1
To determine if the SAG suture synostosis caused by disruption of Axin2
is mediated through endochondral ossification, we examined three critical events in the double-mutant SAG sutures at P10, P15, and P20: chondrocyte resorption, vascular invasion, and osteoblast differentiation, which progressively lead to deposition of a bony matrix. At P10 in the double-mutant animals, the SAG suture consists of a single mesenchymal interface between two adjacent skull bones (), which is also present in the SAG suture of wild-type animals (fig. S1
), and a region beneath the mesenchymal interface where the ectopic chondrocytes are located (), which is not present in the wild-type SAG sutures. By P10 the region of ectopic chondrocytes was comparable to that detected at P7 (). No ectopic chondrocytes were detected at P15 and P20 () and apoptosis, detected by TUNEL staining, in the surrounding area correlated with the reduction of chondrocytes at these stages (), consistent with chondrocyte resorption. Vascular invasion, detected by immunostaining for the endothelial marker laminin, was evident at the site of ectopic chondrogenesis and the area where the chondrocytes had been resorbed (). Whereas at P10, osteoblast differentiation (shown by alkaline phosphatase activity) was detected only at the osteogenic fronts and the periphery of the mineralized bone matrix (), after chondrocyte resorption and vascular invasion, osteoblast differentiation gradually increased in the region below the osteogenic front () and continued to occur at the periphery of the mineralized bony matrix (). These endochondral ossification events are not involved in normal SAG suture development, where only intramembranous ossification at the osteogenic front occurs (fig. S1
). Therefore, ectopic endochondral ossification is a likely mechanism for craniosynostosis that occurs in the Ax2−/−
Fig. 4 Progression of ectopic chondrogenesis to synostosis is mediated by endochondral ossification in the Ax2−/−; Fgfr1+/− SAG suture. Time course study of endochondral ossification is analyzed by AB, TUNEL, immunostaining of laminin, (more ...)
An inverse relation between Axin2 expression and β-catenin activity and FGFR1 abundance exists in the PF suture
Because the SAG suture in the double-mutant mice resembled that of the PF suture in wild-type mice, we examined whether WNT–β-catenin and FGFR1 signaling cooperate in the development of the PF suture with an Axin2GFP
mouse strain (32
) (fig. S2A
) carrying transgenes for the transcription factor rtTA under the control of the Axin2
promoter (Axin2-rtTA) (33
) and a green fluorescent protein (GFP) controlled by tetracycline response elements (TRE-H2BGFP) (34
). With this transgenic system that permits inducible expression of GFP in the presence of doxycycline in cells in which Axin2
is expressed, we monitored the expression of Axin2
in early postnatal development. The Axin2GFP
mice were treated with doxycycline for 3 days before the time of analysis to examine the Axin2
expression pattern. We detected GFP, indicating Axin2
expression, in all cranial sutures at P5 (). In the frontal suture, the intensity of GFP diminished at P7 (), eventually disappearing after P9 (). GFP persisted in all other cranial sutures, indicating that Axin2
expression initially occurs in all cranial sutures and then decreases only in the frontal suture.
Fig. 5 Decreased expression of Axin2 precedes chondrogenesis during PF suture fusion. (A to D) Axin2GFP mice, which are positive for GFP in Axin2-expressing cells, were used to examine the spatiotemporal-specific expression of Axin2 from P5 to P28. Whole-mount (more ...)
We investigated the temporal relation between chondrogenesis and Axin2 expression in the PF suture and found that chondrogenesis was not detectable in the PF suture at P5, the time we detected the greatest amount of Axin2 expression (GFP) in the skeletal precursors located in the suture mesenchyme, osteogenic fronts, and periosteum (). However, Axin2 expression was lower by P9 when chondrogenesis was evident (). In contrast, in the SAG suture where chondrogenesis was not detected, GFP persisted at P9, indicating that Axin2 expression continued in this patent suture ().
The genetic inactivation of the gene encoding β-catenin has a positive effect on chondrogenesis during craniofacial morphogenesis (16
). Because AXIN2 is a negative regulator of WNT signaling and disruption of Axin2
increases β-catenin signaling (14
), areas strongly expressing Axin2
are likely to have low β-catenin activity. We therefore predict that as Axin2
expression decreases in the PF suture, β-catenin signaling should increase. We rarely detected nuclear β-catenin in cells located in the region where the chondrocytes appeared in the PF suture at P5 (), whereas at P7 () and P9 (), β-catenin accumulated in the nuclei of skeletal precursor cells located at the mesenchyme and osteogenic fronts of the PF suture. The decrease in the number of cells expressing Axin2
was accompanied by an increase in the number of SOX9-positive chondrogenic progenitors (). Cells positive for β-catenin were also positive for SOX9, suggesting that β-catenin plays a role in regulation of the SOX9-positive skeletal precursors at the PF suture mesenchyme (). The abundance of FGFR1 was also increased in a temporal and morphological pattern similar to that of β-catenin and SOX9 (), implying that coordinated regulation of these molecules is involved in programming chondrogenesis-mediated PF suture development ().
Genetic ablation of Fgfr1 results in premature chondrogenesis in the PF suture
In the Axin2
mutants, haploid deficiency of Fgfr1
apparently switches the fate of mesenchymal stem cells during suture morphogenesis ( and ). The changes in FGFR1 abundance that occur in the PF suture suggest that this receptor might be involved in chondrogenesis that occurs during PF suture development in early postnatal stages (). FGF2 binds to FGFR1 (35
), and FGF2 signaling has previously been implicated in suture specification (29
). To define the role of FGFR1 in suture development, we created an Fgfr1Ax2
mouse model, in which we integrated tetracycline-dependent activation of a transcription factor produced under the control of the Axin2
promoter and Cre-mediated recombination to ablate Fgfr1
in a spatiotemporal-specific manner in cells that express Axin2
(). With this model, we inactivated Fgfr1
in the Axin2
-expressing cells, including those skeletal precursors at the developing sutures, by treating the mice with doxycycline at birth. We determined the efficacy of this system by crossing mice transgenic for the R26R reporter, which indicates Cre-mediated recombination through the expression of β-galactosidase (β-Gal), into the Fgfr1Ax2
background. The sutures of the resulting Fgfr1Ax2
; R26R skulls were β-Gal–positive, indicating deletion of Fgfr1
(). We confirmed the lack of FGFR1 by immunostaining, which showed the absence of the protein in the Fgfr1Ax2
mutants (). In 80% of the mice, postnatal inactivation of Fgfr1
led to premature chondrogenesis in the PF suture at P7, indicating that Fgfr1
is essential for temporal regulation of chondrocyte development (). However, we did not detect ectopic chondrogenesis in the SAG sutures in the Fgfr1Ax2
mice (). This might be attributed to the difference in Axin2 activity and β-catenin signaling between PF and SAG sutures. In the PF suture where Axin2
expression is low (), β-catenin signaling is high; thus, loss of FGFR1 activity tips the balance toward chondrogenesis. In contrast, AXIN2 is abundant in the SAG suture (); thus, the loss of FGFR1 is not sufficient to change the fate of the mesenchymal stem cells to chondrocytes.
Fig. 6 Targeted disruption of Fgfr1 in the Axin2-expressing skeletal precursors accelerates chondrogenesis in the PF suture. (A) A diagram illustrates ablation of Fgfr1 with a system combining tetracycline-dependent activation and Cre-mediated recombination. (more ...)
Changing the balance of β-catenin and FGFR1 signaling induces mesenchymal cells to form chondrocytes
To confirm that the switch in mesenchymal cell fate observed in the Ax2−/−
mouse skulls was due to an alteration in the balance between β-catenin signaling and FGFR1 signaling, we developed the sβcatAx2
model (fig. S2B
). In this mouse model system, we deleted exon 3 of the gene encoding β-catenin in a spatiotemporal-specific fashion by treating the mice with doxycycline at birth. The resulting truncated β-catenin lacks a phosphorylation site and cannot be targeted for proteasomal degradation. However, neither the SAG suture (fig. S3, A and B
) nor the PF suture (fig. S3, J and K
) of the sβcatAx2
mice exhibited ectopic chondrogenesis. The results indicate that increased β-catenin signaling alone is insufficient to switch the mesenchymal stem cell fate to chondrocytes. Similar to the Ax2−/−
mice, when combined with enhanced β-catenin signaling in the sβcatAx2
mice, haploid deficiency of Fgfr1
resulted in ectopic chondrogenesis in the SAG suture at P7 (fig. S3C
). The SOX9- and COL II–positive chondrogenic progenitors were also evident in the SAG sutures of sβcatAx2
mice, but not those of control or sβcatAx2
mice (fig. S3, D to I
). Thus, both increased β-catenin signaling and decreased FGFR1 signaling appear to be necessary for switching mesenchymal stem cells to the chondrocyte fate.
We explored how the balance of WNT and FGF signaling induced chondrogenesis by analyzing differentiation of primary skeletal precursors isolated from newborn wild-type or Ax2−/−; Fgfr1+/− mouse parietal bones and SAG sutures in culture. Detection of chondrocytes with AB revealed that differentiation of skeletal precursors into chondrocytes was increased in cultures from the Ax2−/−; Fgfr1+/− mice (). The area stained by AB increased ~18 fold in the double-mutant cultures compared to that in the wild-type cultures. In addition, stimulation of β-catenin signaling with BIO, a glycogen synthase kinase 3 (GSK-3) inhibitor, prevented the chondrocyte maturation of primary skeletal precursors isolated from the newborn skulls (). The inhibitory effect of BIO was alleviated by SU5402, an inhibitor of FGFR (). To determine whether the balance of WNT and FGF signaling is a general mechanism regulating chondrogenesis, we isolated primary precursors from bone marrow and performed ex vivo differentiation analysis. The chondrogenic potential was less in the bone marrow cell cultures compared to that in the calvarial cultures. Although the presence of BIO had no significant effects, the addition of BIO and SU5402 increased the formation of AB-positive cells (). Although this is different from the results seen with the cultures of cells isolated from skull bones in which potentiation of β-catenin signaling prevented chondrogenesis, altering β-catenin and FGF signaling still changed the fate of the bone marrow precursors, suggesting that both β-catenin and FGF signaling are important for chondrocyte development in multiple contexts.
Fig. 7 Together, β-catenin and FGF signaling control induction of the chondrocyte fate. In vitro mesenchymal cultures show accelerated chondrogenesis in the Ax2−/−; Fgfr1+/− mutant. (A and B) Primary skeletal precursors, isolated (more ...)
β-Catenin signaling controls the proliferation of skeletal precursor cells
To determine whether the balance of β-catenin and FGF signaling not only controls the differentiation of skeletal precursors, but also controls the proliferation of these precursors, we evaluated the precursor population in the SAG suture in wild-type mice and in mice with mutations that altered β-catenin signaling and FGFR1 signaling, individually or in combination. We detected proliferating cells by immunostaining for Ki67 and phosphorylated histone H3, which are both markers of dividing cells. Although Axin2 deficiency greatly enhanced cell proliferation in the SAG suture, Fgfr1 haploid deficiency had no significant effect (). The number of proliferating cells in the SAG sutures of the Fgfr1Ax2 mice with selective loss of FGFR1 signaling in Axin2-expressing cells was also not different from that in wild-type animals (). Moreover, similar numbers of mitotic cells were detected in the sβcatAx2 and sβcatAx2; Fgfr1+/− mutants (), suggesting that the expansion of precursor population is mediated by the WNT pathway.
Fig. 8 Expansion of skeletal precursors is affected by increased β-catenin signaling, but not by reduced FGF signaling. Sections of the SAG sutures from animals with the indicated genotypes were immunostained for Ki67 or for phosphorylated histone H3 (more ...)
Altering the balance of β-catenin and FGF signaling alters BMP signaling in regions of chondrogenesis
To elucidate the mechanism underlying the β-catenin–FGF–mediated suture morphogenesis, we analyzed the involvement of BMP, a potent chondrogenic stimulator (36
). BMP signaling is increased by Axin2
). In Ax2−/−
SAG sutures, we detected BMP activity in the osteogenic fronts with an antibody that recognizes phosphorylated SMAD1, 5, and 8 (pSMAD1/5/8) (). In the Ax2−/−
mutants, we detected pSMAD1/5/8 in the osteogenic fronts and in the ectopic chondrogenic area () (identified as a region with SOX9-positive cells) (), underlying the suture mesenchyme. These results agree with previous genetic analyses, suggesting that BMP signaling is critical for the chondrocyte activity (38
Fig. 9 BMP signaling is crucial for development of suture mesenchymal stem cells. (A to F) Activation of the BMP pathway and ectopic chondrogenesis coincide in the Ax2−/−; Fgfr1+/− mutants. BMP signaling was detected by immunostaining (more ...)
To determine if FGFR1 signaling affected BMP signaling, we examined BMP activity in the Fgfr1Ax2 model that exhibited accelerated chondrogenesis at the PF suture. Indeed, in the absence of FGFR1 signaling in Axin2-expressing cells, we detected pSMAD1/5/8 in the same region as that containing the SOX9-positive chondrogenic precursors (), suggesting that FGFR1 inhibits the BMP signaling that is involved in chondrocyte differentiation.
To assess the necessity for BMP signaling in the development of mesenchymal stem cells into the chondrogenic lineage in response to altered β-catenin and FGF signaling, we performed an in vitro differentiation analysis with primary skeletal precursors isolated from the skulls of newborn Ax2−/−; Fgfr1+/− mice. Providing additional BMP had a small, but significant, stimulatory effect on chondrocyte maturation in the Ax2−/−; Fgfr1+/− cultures (). The moderate response may be due to the presence of BMP in the cultures, such that the signal is close to maximal already. However, the chondrogenic differentiation of the Ax2−/−; Fgfr1+/− mesenchymal cells was reduced by the addition of the BMP inhibitor Noggin (). Thus, BMP signaling is necessary for the chondrogenesis of mesenchymal skeletal precursors that occurs when the β-catenin–FGF signaling balance is altered.