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Wnt/β-catenin Signaling Regulates Cranial Base Development and Growth
1 Department of Orthopaedic Surgery, Thomas Jefferson University College of Medicine, Philadelphia, PA 19107, USA; Correspondence: * corresponding author, moto{at}dent.asahi-u.ac.jp, eiki.koyama{at}jefferson.edu
Wnt proteins and β-catenin signaling regulate major processes during embryonic development, and we hypothesized that they regulate cranial base synchondrosis development and growth. To address this issue, we analyzed cartilage-specific β-catenin-deficient mice. Mutant synchondroses lacked typical growth plate zones, and endochondral ossification was delayed. In reciprocal transgenic experiments, cartilage overexpression of a constitutive active Lef1, a transcriptional mediator of Wnt/β-catenin signaling, caused precocious chondrocyte hypertrophy and intermingling of immature and mature chondrocytes. The developmental changes seen in β-catenin-deficient synchondroses were accompanied by marked reductions in Ihh and PTHrP as well as sFRP-1, an endogenous Wnt signaling antagonist and a potential Ihh signaling target. Thus, Wnt/β-catenin signaling is essential for cranial base development and synchondrosis growth plate function. This pathway promotes chondrocyte maturation and ossification events, and may exert this important role by dampening the effects of Ihh-PTHrP together with sFRP-1.
Key Words: cranial base synchondrosis • Wnt/β-catenin • sFRP-1 • PTHrP • hedgehog signaling
The cranial base is formed through endochondral ossification and functions as a growth center of the neurocranium. Its central region derives from prechordal, hypophyseal, and parachordal cartilaginous plates that fuse initially to form an uninterrupted cartilaginous structure early in embryogenesis (Thorogood, 1988; Sperber, 2001). Primary ossification centers then emerge, and the cartilaginous segments persisting between the ossification centers represent the synchondroses (Kjaer, 1990; Lieberman and McCarthy, 1999). The synchondroses consist of mirror-image growth plates that display a central reserve zone and zones of chondrocyte proliferation and hypertrophy, and are associated with endochondral and intramembranous bone formation. Given their orientation and location, the synchondroses contribute significantly to anterior-posterior cranial base elongation and overall organization and function (Ingervall and Thilander, 1972; Roberts and Blackwood, 1983; Mooney et al., 1993). Wnt proteins regulate several developmental processes (Nusse and Varmus, 1992). Wnt proteins act by binding to Frizzled and low-density lipoprotein receptor-related receptors on the cell surface, and transduce signals by the highly conserved canonical β-catenin-LEF/TCF pathway (Eastman and Grosschedl, 1999; Huelsken and Birchmeier, 2001) or other pathways (Miller et al., 1999; Veeman et al., 2003). Studies of roles of Wnt signaling in skeletogenesis have shown that Wnt-4, Wnt-5a, Wnt-9a, and Wnt-11 regulate chondrocyte proliferation and differentiation (Hartmann and Tabin, 2001; Church et al., 2002; Spater et al., 2006). We and others have found that the canonical pathway plays dual roles in limb skeletogenesis, where it dampens chondrogenic cell differentiation, but promotes chondrocyte maturation and hypertrophy (Enomoto-Iwamoto et al., 2002; Yang et al., 2003; Day et al., 2005; Tamamura et al., 2005). The canonical pathway is also required for osteogenesis, together with Indian hedgehog (Ihh) signaling (Long et al., 2004; Hill et al., 2005; Hu et al., 2005; Rodda and McMahon, 2006). Wnt antagonist-secreted frizzled-related protein-1 (sFRP-1) is an important negative regulator of Wnt signaling for chondrocyte differentiation and osteogenesis (Wang et al., 2005). Wnt/β-catenin signaling may also have roles in craniofacial skeletal development, as suggested by the phenotype of β-catenin-deficient embryos and cells (Brault et al., 2001; Day et al., 2005). We conducted the present study to determine whether the canonical Wnt/β-catenin signaling pathway specifically regulates growth plate function in mouse cranial base synchondroses. To do so, we used a genetic approach and analyzed mouse embryos conditionally deficient in β-catenin or expressing a constitutive-active form of Lef-1 (CA-LEF1) in developing cartilage.
Generation of Conditional β-catenin Ablation and Transgenic Mice Animal protocols were approved by the IACUC. Mice conditionally deficient in β-catenin were created by mating β-catenin floxed mice (β-cateninfl/fl) possessing loxP sites in introns 1 and 6 of the β-catenin gene (6.129-Ctnnb1tmKem/KnwJ line, purchased from the Jackson Laboratory, Bar Harbor, ME, USA) with Col2a1-Cre mice (kindly provided by Dr. Y. Yamada, NIDCR, Bethesda, MD, USA). Genotyping was performed by PCR with the following primers: fl-β-catenin forward, 5'-AAGGTAGAGTGAT GAAAGTTGTT-3'; fl-β-catenin reverse, 5'-CACCATGTCCTCTGTC TATTC-3'; Cre- forward, 5'-AGACGGAAATCCATCGCTCG-3'; and Cre-reverse, 5'-CCACGACC AAGTGACAGCAATG-3'. To verify Cre specificity, we mated Col2a1-Cre mice with ROSA Cre reporter (R26R) mice and evaluated them for LacZ activity at E15.5 and E17.5 by histochemistry. In total, over 10 wild-type and mutant cranial bases were studied microscopically, and statistical analyses were performed by one-way ANOVA with Tukey’s post hoc comparisons. Mice expressing CA-LEF-1 were prepared as described previously (Tamamura et al., 2005).
Micro-computed Tomography
X-gal Staining
Gene Expression Analysis
Cranial Base and Synchondrosis Abnormalities in β-catenin-deficient Mice First we compared the cranial bases in E17.5 β-catnfl/fl;Col2  1-Cre embryos (heretofore termed β-catenin-deficient) and β-catnfl/wt;Col2a1-Cre and β-catnfl/fl embryos (control) by anatomical inspection and µCT. Control cranial bases displayed a typical elongated morphology, and their pre-sphenoidal (ps), basi-sphenoidal (bs), and basi-occipital (bo) bones and their intrasphenoidal (is) and spheno-occipital (so) synchondroses were also well-defined (Fig. 1A ). Histochemistry showed the E17.5 cranial base bone anlagen to be ossified and stained with alizarin red (Figs. 1B, 1C). In contrast, the cranial bases in β-catenin-deficient littermates remained largely cartilaginous (Figs. 1K, 1L), and bone formation of the cranial base was poor, as revealed by µCT (Fig. 1J) and alizarin red staining (Fig. 1L). In addition, the antero-posterior length of pre-sphenoidal and basi-sphenoidal bones was about 20–25% shorter in mutants than in controls (Figs. 1C, 1L; solid horizontal bar; p < 0.05), while occipital bone length was not markedly affected (Figs. 1C, 1L; dashed horizontal bar). These changes likely contributed to the significant overall antero-posterior shortening of mutant cranial bases.
Growth Plate Organization and Gene Expression in β-catenin-deficient Synchondroses Control E17.5 growth plates displayed a characteristic histology and organization (Fig. 1G  ), with a resting zone with small chondrocytes (rz), a proliferative zone with flat chondrocytes (pz), pre-hypertrophic and hypertrophic zones with columnar chondrocytes (phz and hz), and primary bone spongiosa and marrow. Transcripts for cartilage-characteristic collagen II were present in most zones (Fig. 1D), collagen X transcripts were in the hypertrophic zone (Fig. 1E), and metalloprotease 13 (Mmp-13) transcripts were in the post-hypertrophic zone and bone and marrow cells (Fig. 1F). In contrast, E17.5 β-catenin-deficient growth plates lacked well-defined zones and contained round-to-oval chondrocytes broadly distributed along the entire cranial base (Fig. 1P). Collagen II transcripts were as broadly distributed (Fig. 1M), and collagen X and Mmp-13 transcripts characterized pockets of maturing and hypertrophic chondrocytes (Figs. 1N, 1O). A periosteal bone collar normally forms in tissues flanking the pre-hypertrophic/hypertrophic zones of growth plates and is induced by Ihh (Koyama et al., 1996; Nakamura et al., 1997; St-Jacques et al., 1999). In good agreement, both collagen I (an abundant bone matrix component) and osterix (a bone master regulator) were strongly expressed by the thick perichondrial tissues flanking control spheno-occipital synchondroses (Figs. 1G–1I, arrowheads). However, both markers and ensuing perichondrial bone formation were reduced in β-catenin-deficient synchondroses (Figs. 1P–1R, arrowheads), and this failure was associated with a significant thinning of perichondrial tissues (Fig. 1P, arrowhead). To verify that Wnt/β-catenin signaling was active in developing synchondroses, we used Wnt/β-catenin LacZ-reporter mice and found that reporter activity was mostly appreciable in pre-hypertrophic and hypertrophic zones (Fig. 1S), and that there was also some activity in perichondrial cells (Fig. 1S, arrowheads).
Accelerated Chondrocyte Maturation in CA-LEF1 Transgenic Mice
Altered Hedgehog and Wnt Signaling Patterns in β-catenin-deficient Synchondroses We next focused our attention on the Ihh-PTHrP signaling axis. In controls, Ihh was expressed in the pre-hypertrophic zone (phz) (Figs. 3A, 3B ), and PTHrP in resting and proliferative zones (Fig. 3D). Patched-1 was expressed in the proliferative zone (Fig. 3C, arrows), identifiable by the mitotic marker histone 4C expression (Fig. 3E, arrows), as well as in perichondrial tissues flanking pre-hypertrophic and hypertrophic zones, where perichondrial ossification initiates (Fig. 3C, arrowheads). Notably, endogenous β-catenin transcripts were most evident in proliferative zones and also in perichondrial tissues adjacent to pre-hypertrophic/hypertrophic zones (Fig. 3F, arrowheads). This pattern was virtually super-imposable onto that of Patched-1 (Fig. 3C), suggesting that both Patched-1 and β-catenin are expressed in Ihh target cells. In other systems, the Wnt signaling antagonist sFRP-1 is up-regulated in hedgehog target cells and represents a transcriptional target of hedgehog signaling (Wang et al., 2005; He et al., 2006). In good agreement, sFRP-1 transcripts were obvious in resting and proliferating zones (Fig. 3G) and were abundant along the perichondrial tissues (Fig. 3G, arrowheads).
In β-catenin-deficient synchondroses, expression of Ihh, Patched-1, and PTHrP was low and scattered (Figs. 3I–3K ), as were histone H4C-positive proliferating chondrocytes (Fig. 3L). β-catenin expression was markedly reduced within the growth plate, attesting to the effectiveness of Col2a1-Cre (Fig. 3M), but was also much lower in the perichondrium/periosteum, possibly reflecting diminished local Ihh signaling activity. In line with this possibility, we observed only residual sFRP-1 expression scattered throughout cartilaginous tissue and adjacent perichondrium (Fig. 3N).
Given the above results, one would predict that Wnt signaling and related molecules should not be expressed in mutant synchondroses lacking Ihh. Indeed, in E17.5 Ihh–/–synchondroses, β-catenin gene expression was very low and broad (Fig. 3T
In this study, we provide evidence that the canonical Wnt/β-catenin signaling pathway plays important roles in cranial base synchondrosis development, growth, and function. Deficiency in β-catenin expression in cartilage caused serious defects in chondrocyte proliferation, hypertrophy, and endochondral ossification. Most of the targeted chondrocytes remained small and round, which is characteristic of resting chondrocytes, and failed to become organized in typical growth plate zones. In addition, the β-catenin-deficient growth plates lacked a thick and multilayered perichondrial/periosteal tissue, where osteoprogenitor cells and vascular endothelial cells normally reside. The presence of such altered tissue is associated with, and may have caused, failure of bone collar formation. In reciprocally manipulated synchondroses in which Wnt/β-catenin signaling was constitutively active, chondrocytes underwent precocious maturation and hypertrophy, albeit topographically disorganized, that resulted in intermingling of chondrocytes at different stages of maturation; there was also excessive bone collar formation all along the growth plate flank. Analysis of the data, together, indicates that the canonical Wnt/β-catenin signaling pathway regulates chondrocyte behavior and function in developing synchondroses, and that a tight regulation of this signaling pathway activity is needed for, and facilitates, a normal progression of the chondrocyte maturation process and physiologic synchondrosis function. The synchondrosis growth plates contain distinct zones of chondrocyte maturation and act as the growth centers for cranial bone elongation and expansion during embryonic and post-natal life. As pointed out above, a well-recognized mechanism of growth plate function studied in developing long bones is the PTHrP-Ihh axis, in which Ihh signaling regulates PTHrP expression in peri-articular tissues, and, in turn, PTHrP regulates the rates of chondrocyte proliferation and maturation (Lanske et al., 1996). Recently, we reported that Ihh gene ablation in mouse embryos causes down-regulation of PTHrP expression in the synchondrosis resting zone, reduces chondrocyte proliferation, and leads to widespread hypertrophy, suggesting that the PTHrP-Ihh axis has roles in synchondroses as well (Young et al., 2006). Analysis of the data presented here reaffirms this conclusion, and also shows that functioning of this regulatory axis relies on Wnt/β-catenin signaling. The β-catenin-deficient synchondrosis chondrocytes exhibit poor and deficient expression of PTHrP, Patched-1, and sFRP-1. Since the latter two factors are targets of hedgehog signaling, analysis of the data implies that hedgehog signaling and action are impaired in β-catenin-deficient synchondroses. Analysis of these data also indicates that canonical Wnt signaling has pleiotropic roles in growth plate function, regulates and coordinates a multiplicity of key events, and may do so via interactions with, and modulation of, the PTHrP-Ihh axis. It should be noted here that the phenotype of β-catenin-deficient synchondrosis growth plates is more severe in terms of defects of ossification and gene expression than that of β-catenin-deficient long bones (Day et al., 2005). It is not clear at the moment whether this reflects a greater reliance of synchondrosis growth plates on Wnt/β-catenin signaling and PTHrP-Ihh axis. One of the most salient observations in our study was the considerable accumulation of immature, small, and collagen II-expressing chondrocytes that occupied the bulk of the β-catenin deficient synchondroses. This is in sharp contrast to the situation in CA-LEF transgenic or Ihh–/– synchondroses. Analysis of the data reaffirms the concept that normal organization of growth plates and rates and topology of the chondrocyte maturation process are maintained by fine balances among mechanisms with opposing effects—that is, mechanisms that favor and mechanisms that oppose the unfolding of this process. Chief among known mechanisms that dampen the rate of chondrocyte maturation is the PTHrP-Ihh axis itself (Lanske et al., 1996). Interestingly, however, both the retardation of chondrocyte maturation seen in the β-catenin-deficient synchondroses and the acceleration seen in the Ihh–/– synchondroses are accompanied by a marked decrease in PTHrP expression, signifying that the PTHrP-Ihh axis alone cannot account for both phenotypes, and that other, more complex, mechanisms must be invoked.
Accordingly, then, we propose the following model (Fig. 4
These exciting and plausible scenarios are being tested in ongoing studies.
We express our gratitude to Dr. Y. Yamada for the Col2a1-Cre mice. This work was supported by NIH grants. Received for publication July 5, 2007. Revision received October 30, 2007. Accepted for publication November 27, 2007.
Journal of Dental Research, Vol. 87, No. 3,
244-249 (2008) This article has been cited by other articles:
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