- Research article
- Open Access
β-catenin/cyclin D1 mediated development of suture mesenchyme in calvarial morphogenesis
© Mirando et al; licensee BioMed Central Ltd. 2010
- Received: 15 April 2010
- Accepted: 26 November 2010
- Published: 26 November 2010
Mouse genetic study has demonstrated that Axin2 is essential for calvarial development and disease. Haploid deficiency of β-catenin alleviates the calvarial phenotype caused by Axin2 deficiency. This loss-of-function study provides evidence for the requirement of β-catenin in exerting the downstream effects of Axin2.
Here we utilize a gain-of-function analysis to further assess the role of β-catenin. A transgenic expression system permitting conditional activation of β-catenin in a spatiotemporal specific manner has been developed. Aberrant stimulation of β-catenin leads to increases in expansion of skeletogenic precursors and the enhancement of bone ossification reminiscent to the loss of Axin2. The constitutively active signal promotes specification of osteoprogenitors, but prevents their maturation into terminally differentiated osteoblasts, along the osteoblast lineage. However, the prevention does not interfere with bone synthesis, suggesting that mineralization occurs without the presence of mature osteoblasts. β-catenin signaling apparently plays a key role in suture development through modulation of calvarial morphogenetic signaling pathways. Furthermore, genetic inactivation of the β-catenin transcriptional target, cyclin D1, impairs expansion of the skeletogenic precursors contributing to deficiencies in calvarial ossification. There is a specific requirement for cyclin D1 in populating osteoprogenitor cell types at various developmental stages.
These findings advance our knowledge base of Wnt signaling in calvarial morphogenesis, suggesting a key regulatory pathway of Axin2/β-catenin/cyclin D1 in development of the suture mesenchyme.
- Intramembranous Ossification
- Craniofacial Skeleton
- Bone Ossification
- Osteogenic Front
- Skeletal Precursor
The mammalian skull consists of neurocranium and viscerocranium that are formed from skeletogenic mesenchyme derived from both mesoderm and neural crest . The cranial skeletogenic mesenchyme mainly undergoes intramembranous ossification to form the bone plates . This process differs from endochondral ossification in the appendicular and axial skeletons, which prior formation of cartilage templates is required. The growth of calvaria is then able to accommodate expansion of the brain . During calvarial morphogenesis, cranial sutures serve as skeletogenic growth centers where undifferentiated stem cells develop into calvarial osteoprogenitors, which differentiate to become pre-osteoblasts/osteoblasts localized to the osteogenic front and periosteum. The mature cells then deposit and mineralize bone matrix. Osteoblasts either die by apoptosis or are embedded in the matrix, becoming osteocytes.
A patent suture is necessary for continuous growth of the skull bones. Defects in either cell proliferation, differentiation, or apoptosis affecting the process of intramembranous ossification have been shown to induce premature fusion of cranial sutures, leading to development of craniosynostosis . Abnormalities in the developing suture cause severe malformations of the skull, leading to disruption of brain development. Cranial dysmorphism resulting from suture closure defects can be familial or sporadic in origin [3–5]. Although linkage analyses have shown that genetic mutations are associated with synostosis-related syndromes, the mechanism underlying suture development remains largely elusive. It remains a challenge to understand the maintenance of suture patency which may require undifferentiated cells to be present. These naïve cells are thought to be localized in the suture mesenchyme. Better understanding of the genetic regulatory network conveying signals to orchestrate the suture morphogenetic processes promises important insights into pathogenesis of congenital deformities.
Our previous work has linked an evolutionary conserved Wnt signal transduction pathway for the first time to craniosynostosis . Mice with genetic inactivation of Axin2, a negative regulator targeting β-catenin degradation, exhibit suture abnormalities. The synostosis phenotype is caused by accelerated intramembranous ossification mediated through a dual role of β-catenin in both the expansion of osteoprogenitors and the maturation of osteoblasts [6, 7]. Moreover, haploid deficiency of β-catenin alleviates the premature closure and calvarial osteoblast defects. The loss-of-function analysis thus suggests the requirement of β-catenin in Axin2 mediated calvarial morphogenesis.
In this study, we employ a gain-of-function analysis to determine if aberrant activation of β-catenin induces calvarial defects reminiscent to the Axin2 mutant. A transgenic system integrating the tetracycline-dependent activation and Cre-mediated recombination methods has been developed. Our findings demonstrate that inducible stimulation of β-catenin causes severe suture abnormalities resembling those with the Axin2 ablation. The expansion of skeletogenic precursors and their subsequent differentiation are altered, leading to increased bone ossification. β-catenin signaling plays a critical role in development of the suture mesenchyme through modulation of calvarial morphogenetic signaling pathways. Furthermore, analyzing mice with disruption of the β-catenin transcriptional target, cyclin D1, we reveal its essential role in the expansion of skeletal precursors. Cyclin D1 is required for propagation of specific types of osteoprogenitors at various developmental stages.
We then developed a transgenic system combining the tetracycline-dependent activation and Cre-mediated recombination methods to determine if aberrant activation of β-catenin affects suture development (Figure 1G). This advanced system permits inducible stimulation of β-catenin during calvarial morphogenesis. It allows us to test if constitutive activation of β-catenin signaling causes suture abnormalities, leading to defects in skeletogenesis, similar to that observed in the Axin2 nulls. First, mice carrying the Axin2-rtTA transgene were crossed with the TRE-Cre mice to obtain the Axin2Cre model. Next, the β-catΔEx3Fx allele was introduced to the Axin2Cre mice. In these triple transgenic animals, Cre recombinase was produced in the Axin2-expressing cells in the presence of Dox. The expressed Cre then deleted exon 3 of β-catenin to generate a truncated protein. As a result, a stabilized dominant protein (sβ-cat), which lacks important phosphorylation sites  and cannot be targeted for proteosome degradation, was generated in the Axin2-expressing cells (Figure 1G). This sβcatAx2 mouse model permits stimulation of β-catenin in a spatiotemporal specific fashion during formation of the craniofacial skeleton. Inducible expression of sβ-cat in the Axin2-expressing cells had significant effects on craniofacial skeletogenesis (Figure 1H-J). The suture regions of the sβcatAx2 mice were drastically expanded (Figure 1K-M). In addition, bone ossification was enhanced in these mutants compared to the control (Figure 1N-P).
Our findings demonstrated that stimulation of β-catenin in the skeletogenic mesenchyme promotes expansion of skeletal precursors, specification of osteoblast cell types, maturation of matrix-producing osteoblasts, and FGF and BMP signaling. These phenotypic and regulatory defects, reminiscent to the Axin2 deficiency, thus provide evidence to support an essential role of β-catenin in mediating the Axin2-null phenotypes using a gain of function approach.
Further examination revealed that cyclin D1 deficiency reduced both Runx2 negative and positive populations at E17.5 (Figure 7H, I, L, M). However, at newborn, the expression of Ki67 (Figure 7J, N) remained affected at the suture mesenchyme, but became unaffected at the osteogenic fronts of cyclin D1-/- which are Runx2-expressing cells (Figure 7K, O). This implies that the cyclin D1 ablation interferes with the expansion of naïve cells in the developing suture at early developmental stages. As the development proceeds, the effects on the committed osteoprogenitors were somehow alleviated. At newborn, we also observed the status of FGF and BMP signaling in the skeletal precursors at the osteogenic fronts of cyclin D1-/-, which were comparable to the wild type counterparts (Additional file 1 Figure S1). The skeletogenic activities at the mutant osteogenic fronts appeared to be restored at newborn.
Studies in the past have suggested the importance of β-catenin signaling in modulating osteoblast proliferation and differentiation in health and disease [16–23]. It has been previously proposed that proliferation of skeletal precursors requires β-catenin [16, 18]. In the subsequent development of osteoblasts, β-catenin needs to be activated [16–18]. Our prior study of mice with disruption of Axin2 also indicates a dual role of β-catenin in expansion of skeletal precursors and their differentiation into osteoblasts . There is no question about the essential function of β-catenin in regulating the expansion of skeletal precursors. However, there is a need to obtain additional in vivo evidence for its involvement in the maturation process. This has been an obstacle due to the effect of cell proliferation also interfering with cell differentiation. Indeed, both deletion and stimulation of β-catenin in the skeletogenic mesenchyme result in delay of calvarial development at embryonic stages [16, 17].
Using sophisticated mouse genetic systems, we are able to further our investigation by manipulating the activity of β-catenin in the Axin2-expressing cells within the suture mesenchyme. This study provides direct in vivo evidence to support that β-catenin regulates both proliferation and differentiation of osteogenic precursors. Stimulation of β-catenin with transgenic expression of a dominant mutant protein in the suture mesenchyme results in calvarial deformities. In these mutants, the expansion of skeletal precursors and their differentiation into osteoblasts are highly stimulated. These abnormalities are reminiscent to those observed in the Axin2 mutants. The gain-of-function study strongly supports the hypothesis for a dual role of β-catenin in cell proliferation and differentiation during osteoblast development [6, 7].
The sβcatAx2 mice do not seem to display the exact same phenotype as the Axin2 mutants. Axin2, working upstream of β-catenin in the canonical Wnt pathway, might possess additional functions affecting other signaling molecules not regulated by β-catenin. Alternatively, the presence of Axin1 might compensate for the loss of Axin2 in a certain degree as these two genes can functionally substitute for each other . While Axin1 is still able to regulate β-catenin in the Axin2 mutants, the stabilized form of β-catenin can no longer be modulated by Axin proteins in the sβcatAx2 mice resulting in the phenotypic difference between the two models.
Because of our model permitting conditional stimulation of β-catenin, we have been able to overcome the embryonic lethality and examine its role in postnatal development of the craniofacial skeleton. In early juveniles, the calvarium grows rapidly while skeletal precursors are required to expand significantly. Alterations of β-catenin at this stage cause severe proliferation and differentiation defects. In contrast, the skeletal precursor pool is relatively quiescent after mice are 3 weeks old. In late juveniles, expansion of skeletal precursors does not seem to be affected by aberrant β-catenin signaling while high levels of bone ossification are detected (Mirando and Hsu, unpublished). Unfortunately, mice with inducible expression of the dominant β-catenin protein are unable to survive beyond 4 weeks after the initial activation of transgene takes place. The lethality issue, most likely due to the Axin2 promoter being active in other tissues and organs, has prevented us from assessing the long term effect of the transgenic expression. Nonetheless, the new finding nicely complements our previous work using a loss-of-function analysis in which haploid deficiency of β-catenin alleviates the skull defects caused by the Axin2 disruption .
We have previously shown that as a transcriptional target of Wnt, cyclin D1 is modulated by alterations of the Axin2/β-catenin regulatory pathway [6, 7]. However, there is a lack of information on linking the cyclin D1 function to skeletogenesis [28, 29]. To assess the role of cyclin D1 in calvarial morphogenesis, a genetic study was performed. Cyclin D1 apparently has an essential function in calvarial development at embryonic stages. In the cyclin D1 knockout embryos, the expansion of both Runx2 negative and positive skeletal precursor populations are impaired at the suture mesenchyme and osteogenic fronts, respectively. Deficiencies in cell proliferation and differentiation have been shown to cause ossification delay of the skull [30, 31]. We have identified similar types of ossification deficiencies in the cyclin D1 knockouts. The finding thus provides first genetic evidence for the involvement of cyclin D1 in calvarial morphogenesis (Figure 9).
As a direct transcriptional target of β-catenin, cyclin D1 acts in this regulatory pathway. However, the loss of cyclin D1 interferes with only the Runx2 negative naïve cells within the suture mesenchyme, but not the Runx2 positive progenitors at the osteogenic fronts at newborns. The skeletogenic signaling pathways of FGF and BMP are also unaffected at the osteogenic fronts. Our data imply that the restoration of skeletogenic activities at the osteogenic fronts of cyclin D1-/- might be a compensation effect of other cyclins with similar function. The loss of cyclin D1 may be compensated by cyclin D2, D3 and E. Nevertheless, we cannot rule out the possibility that the lethality associated with the cyclin D1 deletion could result in selected phenotypic representation. This is because only 72% of mice with disruption of cyclin D1 able to survive after birth . The mutants with most severe phenotypes are unable to be obtained due to abnormalities in other tissues and organs. Furthermore, our study of cyclin D1 in craniofacial skeletogenesis raises a question on its involvement in development of the axial and appendicular skeletons. It remains to be determined whether the D-type cyclin(s) has a role in other developments and maintenance of the body skeleton.
Using sophisticated mouse genetic studies, we demonstrate that aberrant stimulation of β-catenin causes calvarial defects resembling those of the Axin2 mutation. Constitutive activation of β-catenin enhances both expansion of skeletogenic precursors and bone ossification. Together with our previous findings, these results indicate that β-catenin plays an essential role in the calvarial morphogenetic signaling pathways. In addition, as a direct transcriptional target of β-catenin, cyclin D1 is required for the expansion of skeletal precursors during calvarial morphogenesis. This study provides genetic evidence to further support the importance of Wnt signaling in calvarial morphogenesis, suggesting a key regulatory pathway of Axin2/β-catenin/cyclin D1 in development of the suture mesenchyme.
The Axin2-rtTA [32, 33], TRE-lacZ [34, 35], TRE-H2BGFP [8, 33, 34], TRE-Cre , β-catΔEx3Fx , cyclin D1  mouse strains and genotyping methods were reported previously. To test the Dox-inducible expression system, mice carrying the Axin2-rtTA and TRE-lacZ transgenes were obtained and treated with Dox (2 mg/ml plus 50 mg/ml sucrose) for several days as described [8, 32, 34, 35]. For generating the sβcatAx2 mouse strain, mice carrying Axin2-rtTA and TRE-Cre transgenes were first bred into the β-catΔEx3Fx heterozygous background . The expression of a degradation-deficient β-catenin in the Axin2-expressing cells was then induced by Dox treatment at E16.5 or E17.5. Care and use of experimental animals described in this work comply with guidelines and policies of the University Committee on Animal Resources at the University of Rochester.
Isolation and culture of primary skeletal precursors were performed as described . Briefly, calvarial cells isolated from newborns were cultured in αMEM media containing 10% fetal calf serum. Only the first passage cells were used for the study. To induce the expression of the transgene, Dox was added to the culture media for 8 hours. Cells (1 × 104) were seeded in 24 well plates, and maintained in differentiation media containing 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate for 6 days, followed by alkaline phosphatase staining as described .
Histology, skeletal preparation, β-gal staining and GFP analysis
Skulls were fixed for skeletal preparation or paraffin embedded, sectioned and stained with hematoxylin/eosin for histology or von Kossa staining as described [6, 7]. Details for β-gal staining in whole mounts or sections were reported previously [32, 34, 35]. For analysis in sections, newborn skulls were further fixed in 4% PFA/PBS, processed for frozen section, and evaluated by histology using Zeiss Axio Observer microscope (Carl Zeiss, Thornwood, NY).
Immunological staining with avidin:biotinlylated enzyme complex was performed as described [6, 7]. The staining was visualized by enzymatic color reaction or fluorescence according to the manufacture's specification (Vector Laboratories, Burlingame, CA). Images were taken using Zeiss Axio Observer microscope. Mouse monoclonal antibodies Cdk4 (Cell Signaling, Danvers, MA), BrdU (Thermo Fisher, Fremont, CA), Runx2 (MBL International, Woburn, MA), Osteopontin (Developmental Studies Hybridoma Bank, Iowa City, IA), cyclin D2 (Thermo Fisher), cyclin D3 (Thermo Fisher), cyclin E (Santa Cruz) and ABC (recognizing activated/non phosphorylated form of β-catenin, Upstate; Billerica, MA); rabbit polyclonal antibodies cyclin D1 (Thermo Fisher), Fgfr1 (Santa Cruz, Santa Cruz, CA), Fgfr2 (Santa Cruz), Fgf2 (Santa Cruz), Ki67 (Thermo Fisher), Osterix (Abcam, Cambridge, MA) and phospho-Smad1/5/8 (Cell Signaling) were used in these analyses. For statistical evaluation, the stained images were taken to determine the percentage of positive cells by counting the positively stained cells in a total population of cells (positive plus negative). The counting process was repeated in different samples for a number of times (n represents the number of animals or cell cultures) to obtain the mean values and error bars. The p values were obtained using student t test.
We thank Hartmut Land for reagents, Brian H. Son for technical assistance. This work is supported by National Institutes of Health grant DE15654 to W.H.
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