Osteoporosis is characterized by reduced bone mass along with microarchitectural deterioration of the skeleton, increasing the risk of fragility fractures.1 In osteoporosis, bone mineral density (BMD) is reduced as a result of an imbalance in bone formation and resorption. Because of the strongly heritable nature of BMD and bone geometry, genes involved in the regulation of BMD as well as BMD-associated loci have been described.2–6 Three of those genes, CTNNB1, LRP5, and Gpr177, are intimately involved in the Wnt signal transduction pathway.3–5, 7
β-catenin, encoded by CTNNB1, is a master regulator for transducing the canonical Wnt pathway.8, 9 It has been well established that Wnt/β-catenin regulates bone formation and remodeling through modulation of osteoblasts and osteoclasts.10–13 In development of skeletogenic mesenchyme, the upstream as well as downstream effectors of β-catenin, including Axin2 and cyclin D1, are tightly associated with calvarial morphogenesis in health and disease.14–17 Genetic studies have shown that not only is osteoblastogenesis causally affected by alteration of Wnt/β-catenin signaling,14–15, 17 but its interplay with fibroblast growth factor (FGF) and bone morphogenic protein (BMP) also determines the fate of stem cells.16LRP5, encoding a Wnt receptor, has been strongly implicated in the regulation of bone mass.8, 9, 18 In humans, loss-of-function mutations in LRP5 are genetically linked to osteoporosis-pseudoglioma syndrome, characterized by low bone density and skeletal fragility.19 In contrast, gain-of-function mutations result in high bone mass.20 Successful development of mouse models, mimicking the observed phenotypes, further demonstrates that LRP5 controls bone formation through modulation of osteoblast proliferation.21
We have recently shown that Gpr177 is the mouse orthologue of Drosophila Wls (also known as Evi and Srt), whose gene product is essential for Wnt sorting and secretion.22–24 Disruption of Gpr177 in mice causes defects in patterning of the embryonic anterior-posterior axis, a phenotype highly reminiscent of the loss of Wnt3.22 The Wnt3 null phenotype is the earliest abnormality found in all Wnt knockouts, suggesting that the Gpr177-mediated regulation of Wnt cannot be substituted. As a transcriptional target of Wnt, Gpr177 is widely expressed during embryogenesis, leading to a hypothesis that reciprocal regulation of Wnt and Gpr177 is required for development of various organs in health and disease.22, 25 However, the actual involvement of Grp177 in these processes, including skeletogenesis, remains unclear. The implication of Gpr177 in human BMD and osteoporosis-related traits prompts us to investigate the importance of Gpr177 in skeletal development. Because of the early lethality associated with the inactivation of Gpr177, a mouse strain permitting its conditional deletion has been created.26 A number of mouse models were generated to determine its role in various skeletogenic cell types. This study not only reveals the requirement for Gpr177 in osteogenesis and chondrogenesis, but also identifies its essential function in modulating the interplay of Wnt signals across distinct cell types in skeletal development.
Materials and Methods
The Gpr177Fx, Dermo1-Cre, Osx-Cre, Col1a1-Cre, and Col2a1-Cre mouse strains and genotyping methods have been reported.26–30 In brief, Dermo1-Cre and Osx-Cre are the Cre knock-in alleles of Dermo1 and Osx. Col1a1-Cre and Col2-Cre are transgenic lines expressing Cre under control of the murine 2.3-kb collagen type I alpha 1 (Col1a1) and murine 6-kb collagen type II alpha 1 (Col2a1) promoters, respectively. 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.
Histology, beta-galactosidase staining, immunostaining, in situ hybridization, and skeletal analysis
Embryos were fixed, paraffin-embedded, and sectioned for histological evaluation as described.31, 32 Details for beta-galactosidase (β-gal) staining in whole mounts and sections, and for skeletal preparation and staining have been described.16, 33 In situ hybridization and immunostaining analyses were performed as described,22, 31, 34, 35 In brief, DNA plasmids, containing Col2a1, Indian hedgehog (Ihh), Col10a1, Col1a1, matrix metalloproteinase 9 (MMP9), MMP13, Runt-related transcription factor 2 (Runx2), Osterix (Osx), and Osteocalcin (OC) cDNAs, were linearized for in vitro transcription using T3 or T7 RNA polymerase (Promega, Wisconsin, WI, USA) to generate digoxigenin-labeled RNA probes for in situ hybridization.36–38 Sections were then incubated with the RNA probes, followed by recognition with an alkaline phosphatase conjugated anti-digoxigenin antibody (Roche, Indianapolis, IN, USA). To visualize the bound signals, samples were incubated with BM-purple (Roche) for 4 to 5 hours. For immunostaining, mouse monoclonal antibodies Runx2 (MBL International, Woburn, MA, USA) and ABC (Millipore, Billerica, MA, USA), rabbit polyclonal antibodies Gpr177,22, 25 Osterix (Abcam, Cambridge, MA, USA), platelet endothelial cell adhesion molecule (PECAM-1) (Santa Cruz, Santa Cruz, CA, USA), and rabbit monoclonal antibody Ki67 (Thermo Fisher, Barrington, IL, USA) were used. Proper controls for in situ hybridization with the sense RNA probes and immunostaining without the primary antibodies are shown in Supplemental Fig. S7. For statistical analysis, the number of proliferating skeletal precursors (Runx2–; Ki67/phosphorylated histone H3 [pHH3] +; 4′,6-diamidino-2-phenylindole [DAPI] + ) is divided by the number of skeletal precursors (Runx2–; Ki67/pHH3 ± ; DAPI +) residing in the calvarial skeletogenic mesenchyme. To examine proliferation of the osteoprogenitors, the number of proliferating osteoprogenitors (Runx2 + ; Ki67/pHH3 + ; DAPI +) is divided by the total number of osteoprogenitors (Runx2 + ; Ki67/pHH3 ± ; DAPI +).
Gpr177 is essential for development of craniofacial and body skeletons
To determine the role of Gpr177 in skeletal development, conditional deletion was performed in various skeletogenic cell types. First, we generated Gpr177Dermo1 mutant mice in which Gpr177 was inactivated by the Dermo1-Cre transgene in the mesenchymal cells.30 The mesenchymal deletion of Gpr177 severely impaired development of the craniofacial skeleton at embryonic day 15.5 (E15.5). Alizarin red and Alcian blue staining showed that formation of the calvarial, maxillary, and mandibular bones mediated by intramembranous ossification is defective or completely missing in the Gpr177Dermo1 embryos (Fig, 1A, D). Mineralization of the frontal bone was not detected in the mutants (Supplemental Fig. S1A, B). Because Dermo1-Cre induces recombination in the mesenchymal cells, Gpr177 is ablated not only in the precursors but also in their osteoblast derivatives.39, 40 To further our assessment of the requirement of Gpr177 in the osteoprogenitors and osteoblasts, we generated Gpr177Osx and Gpr177Col1 mutants, in which Gpr177 was inactivated by Osx-Cre29 and Col1a1-Cre,27 respectively. Compared to the control littermates, no obvious defect in development and mineralization of the craniofacial bones was detected in either of the mutants at E15.5 (Fig. 1B, C, E, F and Supplemental Fig. S1C–F). At birth, the Gpr177Osx calvaria seemed to display delayed mineralization (Supplemental Fig. S2A–C). However, this is most likely caused by the Cre transgene, but not the Gpr177 deletion, because similar effects were also shown in the Osx-Cre; Gpr177 + /+ calvaria (Supplemental Fig. S2A–C). Moreover, the Gpr177Col1 calvaria did not show any deformities, confirming that Gpr177 is dispensable in the osteoblasts (Supplemental Fig. S2D–F). The use of the R26R allele ensured the effectiveness of Cre lines (Supplemental Fig. S3A–D). Immunostaining of Gpr177 also revealed its loss of expression in the expected regions, including the skeletogenic mesenchyme and osteogenic front (Supplemental Fig. S3E–L). Because the deletion of β-catenin by Osx-Cre severely impairs calvarial development,29 the analysis of Gpr177 may provide new insight into the cell type responsible for Wnt production and signaling during intramembranous ossification. Although dispensable in the Osx-expressing osteoprogenitors and Col1-expressing osteoblasts cells, Gpr177 plays an important role in the mesenchymal cells essential for intramembranous ossification during calvarial development.
To determine the role of Gpr177 in endochondral ossification, we examined the formation of the appendicular long bones in the Gpr177Dermo1 mutants. The mesenchymal deletion of Gpr177 causes severe defects in development of the body skeleton, including forelimbs and hindlimbs at E15.5 (Fig. 1G–L). Mineralization occurred in the collar bones and primary spongiosa of control littermates, but was missing in the Gpr177Dermo1 mutants (Supplemental Fig. S1G, H). This is also accompanied by the delay of chondrocyte maturation, because hypertrophic chondrocytes were almost undetectable at this stage (Supplemental Fig. S1I, J). To examine whether the presence of Gpr177 in the mesenchymal cells is sufficient for chondrogenesis, we generated Gpr177Col2 mutants in which Gpr177 was inactivated by the Col2a1-Cre transgene in chondrocytes.28 The removal of Gpr177 in the chondrocytes caused defects in the formation of axial and appendicular bone (Fig. 1M, P). In the E15.5 Gpr177Col2 mutants, the long bones were shortened and bone matrix formation was dramatically affected (Fig. 1N, O, Q, R). The chondrogenic deletion of Gpr177 significantly reduced bone mineralization, and interfered with chondrocyte maturation (Supplemental Fig. S1K–N). Therefore, it is necessary to have Gpr177 present in the chondrocytes, although we cannot rule out its requirement in the mesenchymal cells during endochondral ossification.
The role of Gpr177 in osteoblast development
The development of calvarial bone plates, including the frontal and parietal bones, is mediated by intramembranous ossification.41, 42 At about E12.5, the initial formation of a frontal bone primordium, which is sandwiched between the developing eye and brain, initiates osteogenesis.43 The osteogenic process then extends apically from the skull base to the midline within the skeletogenic mesenchyme, characterized by expression of the osteoprogenitor markers, Runx2 and Osx, and the osteoblast markers, Col1a1 and OC (Fig. 2A–D). In contrast, the expression of these markers was strongly reduced or not detectable in the Gpr177Dermo1 mutants (Fig. 2E–H), suggesting that the mesenchymal expression of Gpr177 is essential for osteoblast differentiation.
To determine if the expansion of osteoblast precursors was also affected by the Gpr177 ablation, cells undergoing mitotic division were detected by immunostaining of Ki67 (Fig. 3A, D) and phosphorylated Histone H3 (Fig. 3G, J). Consistent with our prior observation,17 there are two populations of precursors, Runx2-negative and Runx2-positive, actively expanding in the skeletogenic mesenchyme during intramembranous ossification (Fig. 3A–C, G–I). However, proliferation of these two populations was severely affected in the Gpr177Dermo1 mutants (Fig. 3D–F, J–L). Statistical analysis showed that the Gpr177 deletion significantly reduces the numbers of actively proliferating Runx2-negative and Runx2-positive cells (Fig. 3M, N). The mesenchymal expression of Gpr177 is therefore essential for expansion of precursor cells and their differentiation into osteoblast cell types.
Gpr177 in mesenchymal but not osteoblast cells is necessary for Wnt production in activation of β-catenin signaling during intramembranous ossification
The loss of Gpr177 might affect Wnt signaling during skeletal development. To assess this, the activation of β-catenin and its transcriptional target, Axin2, was examined by immunostaining with the anti-activated form of β-catenin (ABC) and β-gal staining of the Axin2lacZ (Ax2lacZ) knock-in allele, respectively.14, 16, 22, 34 Nuclear expression of β-catenin and uniform activation of Axin2 were evident in the E15.5 skeletogenic mesenchyme (Fig. 4A, D, G, J). The expression of β-catenin and Axin2 was highly diminished in the Gpr177Dermo1 (Fig. 4B, E, H, K), but not the Gpr177Osx (Fig. 4C, F, I, L) mutants. In the Osx-expressing osteoprogenitors, although β-catenin is required for Wnt signal transduction,29 the Gpr177-mediated production of Wnt is apparently dispensable. The mesenchymal cells are the major and essential source of Wnt in osteoblastogenesis during calvarial morphogenesis.
To examine the signal-producing and signal-receiving cells of Wnt, we investigated the expression of Gpr177 and Axin2, respectively. Although the expression of Osx was restricted to osteoprogenitors at the osteogenic front, Gpr177 showed a uniform expression pattern in the skeletogenic mesenchyme (Fig. 4M–R). Therefore, Gpr177 is expressed in the mesenchymal cells and the osteoprogenitors even though their production of Wnt in the osteoprogenitors is not necessary for calvarial development. In agreement with β-catenin being required for both mesenchymal and osteoprogenitor cells,29, 38, 40, 44 we found that canonical Wnt signaling is uniformly activated in the skeletogenic mesenchyme using the Axin2lacZ allele (Fig. 4S–U). Our findings suggest that mesenchymal production of Wnt activates β-catenin signaling in mesenchymal and osteoblast cell types in calvarial bone development.
Gpr177 is required for endochondral ossification
To determine the role of Gpr177 in development of the body skeleton, we performed a comprehensive molecular analysis, examining the key steps of endochondral ossification, including chondrocyte proliferation, chondrogenesis, extracellular matrix (ECM) remodeling, vascular invasion, and osteoblast differentiation. The number of cells undergoing mitotic division is significantly reduced in the columnar zone, but not epiphyses of the Gpr177Dermo1 and Gpr177Col2 humeri, suggesting that expansion of the proliferating and prehypertrophic but not the resting chondrocytes was affected by the loss of Gpr177 (Supplemental Fig. S4). In the Gpr177Dermo1 mutants, the expression of Col2a1 in the chondrocytes was not affected at E15.5 (Fig. 5A, F). Two expression zones of Ihh and Col10a1 separated by the marrow cavity were evident in the control littermates (Fig. 5B, C). However, Ihh and Col10a1 had just begun to be expressed in the center of the Gpr177Dermo1 humerus, suggesting severe delay of chondrocyte maturation (Fig. 5G, H). The expression of MMP9 and MMP13 in the hypertrophic chondrocytes during ECM remodeling45, 46 was also absent in the mutants (Fig. 5D, E, I, J). Vascular invasion, characterized by immunostaining of PECAM-1, also did not occur (Fig. 5K, P). Furthermore, strong expression of Runx2 in the perichondrium, collar bone, and primary spongiosa, as well as in the hypertrophic chondrocytes of control, was diminished significantly in the mutant (Fig. 5L, Q). This was accompanied by decreased expression of Osx and Col1a1 in the collar bone and perichondrium of Gpr177Dermo1 (Fig. 5M, N, R, S). The expression of OC in the bone collar region was not detectable (Fig. 5O, T). These results indicate that the disruption of endochondral ossification starts at chondrocyte maturation, and the subsequent events, including ECM remodeling, vascular invasion, and osteoblastogenesis, are impaired in the Gpr177Dermo1 mutants.
Endochondral ossification requires the presence of Gpr177 in chondrocytes
We then examined the role of Gpr177 in the chondrocytes during long-bone development. A comprehensive molecular analysis was carried out to examine the key steps of endochondral ossification in the E15.5 Gpr177Col2 embryos. Similar to those of Gpr177Dermo1, the expression of Col2a1 remained unchanged whereas the expression of Ihh was greatly reduced in the Gpr177Col2 mutants (Fig. 6A, B, F, G). The marrow cavity and its surroundings, defined by two hypertrophic zones expressing Col10a1, were significantly smaller in the Gpr177Col2 humerus (Fig. 6C, H). In addition, the expression of MMP9, MMP13, and PECAM-1 indicated that ECM remodeling and vascular invasion are defective in the Gpr177Col2 mutants (Fig. 6D, E, I, J, K, P). The expression of Runx2, Osx, and Col1a1 showed that osteoprogenitor cells are dramatically reduced and translocation of osteoblasts from the perichondrium to the nascent primary ossification center did not occur (Fig. 6L–N, Q–S). Furthermore, the mature osteoblasts expressing OC were missing in the Gpr177Col2 mutants (Fig. 6O, T).
At E17.5, bone formation remained severely impaired in the humerus of Gpr177Col2, compared to the control (Supplemental Fig. S5). Collar bones normally extended from diaphysis to the perichondrial region (Supplemental Fig. S5A, B). However, no bone collar was formed in the perichondrial region of Gpr177Col2 although mineralization of the hypertrophic chondrocytes was evident (Supplemental Fig. S5G, H). In addition, chondrogenesis, ECM remodeling, and osteoblastogenesis were severely defective in the mutants (Supplemental Fig. S5C–F, I-X), suggesting that the presence of Gpr177 in the chondrocytes is essential for endochondral ossification. To determine if the presence of Gpr177 in the osteoblasts is required for endochondral ossification, we examined the Gpr177Col1 limbs. Similar to that observed in calvarial development (Fig. 1), the deletion of Gpr177 by Col1a1-Cre also did not cause any abnormality in limb development (Supplemental Fig. S6). Thus, Gpr177 is dispensable in the osteoblasts during intramembranous and endochondral ossifications. Our findings suggest that the impairment of osteoblast differentiation in the Gpr177Dermo1 and Gpr177Col2 limbs is attributed to delay in chondrocyte maturation but not to intrinsic defects of the osteoblasts.
Gpr177 regulates Wnt signaling in long-bone development
We next studied the effect of Gpr177 deficiency on the canonical Wnt pathway during limb development. The expression of Gpr177 was found in the resting, proliferating, and hypertrophic chondrocytes, as well as the in the perichondrium in the developing limb (Fig. 7A–C). Nuclear staining of the activated β-catenin was strong in the resting and proliferating chondrocytes, and in the perichondrium, but very weak in the hypertrophic chondrocytes (Fig. 7D–F). Immunostaining of Gpr177 further showed the effective ablation of Gpr177 in the proliferating and hypertrophic chondrocytes in the Gpr177Dermo1 and Gpr177Col2 mutants (Fig. 7G–I). The reduction of nuclear β-catenin staining further indicated that the Gpr177 deletion disrupts canonical Wnt signaling (Fig. 7J–L). Crossing of the Ax2lacZ allele into the Gpr177Dermo1 and Gpr177Col2 backgrounds revealed that the expression of Axin2 is drastically reduced in the chondrocytes and perichondrium (Fig. 7M–O). The data thus suggest that Gpr177 in chondrocytes is necessary for activation of canonical Wnt signaling during endochondral ossification.
This study provides evidence that Gpr177, a gene closely linked to BMD and osteoporosis-related traits, is required for skeletogenesis. Genetic inactivation of Gpr177 in the mesenchymal cells severely impairs intramembranous and endochondral ossifications. Gpr177 plays an essential role in osteoblastogenesis and chondrogenesis through modulation of cell proliferation and differentiation. In the Gpr177 mutants, Wnt/β-catenin signaling is greatly reduced during development of the calvaria and limbs, suggesting that Gpr177-mediated regulation of Wnt production cannot be substituted. Genetic inactivation of Gpr177 in the osteogenic or chondrogenic progenitors further reveals its distinct role in osteogenesis and chondrogenesis. In osteoblast development, the presence of Gpr177 in the Osx-expressing osteoprogenitors and the Col1-expressing osteoblasts is dispensable because no skull defects associated with its ablation were detected in the mutants. In contrast, mesenchymal production of Wnt mediated by Gpr177 is necessary for intramembranous ossification. However, the deletion of β-catenin by Osx-Cre causes severe skull abnormalities, indicating that canonical Wnt signaling in the Osx-expressing osteoprogenitors is necessary for osteoblastogenesis.29 Our finding suggests that mesenchymal cells are the main cell type responsible for signal production. Their supply of Wnt activates β-catenin signaling in the signal-receiving mesenchymal and osteoblast cells. Both mesenchymal autocrine and paracrine signals of Wnt are essential for osteoblast development (Fig. 7P). For development of the chondrocytes, however, chondrocyte production of Wnt is essential for chondrogenesis, although the mesenchymal supply might also be necessary (Fig. 7P). The chondrocyte-specific deletion of Gpr177 by Col2a1-Cre causes deficiencies in skeletal development, albeit less severe than those of caused by Gpr177Dermo1. Several key steps of endochondral ossification, including chondrocyte proliferation and maturation, ECM remodeling, vascular invasion, and osteoblast differentiation, are delayed in the developing humerus. These defects are reminiscent of those found in the mutants with ablation of β-catenin in the mesenchymal cells or chondrocytes,38, 47 suggesting that Wnt proteins produced by chondrocytes are indispensable for endochondral ossification. Our findings lead us to propose a new mechanism underlying development of the skeleton mediated by the canonical Wnt pathway. The intercross of Wnt signaling between undifferentiated mesenchymal cells and skeletogenic progenitors is well orchestrated in a cell type–specific manner.
Canonical Wnt signaling has been demonstrated to play an important role in skeletogenesis, including the determination of mesenchymal cell fate29, 40, 44 and chondrocyte maturation.38, 47 Based on the molecular analysis of Gpr177Dermo1 and Gpr177Col2 mutants, specification of the Col2a1-expressing chondrocytes does not seem to be affected. However, chondrocyte proliferation and maturation are significantly reduced in both mutants. Our study of Gpr177 therefore supports an important function of β-catenin signaling in chondrogenesis at later stages. Because of ectopic chondrogenesis detected in the total β-catenin loss-of-function mutants, it has been postulated that Wnt signaling possesses an inhibitory effect on chondrogenesis.29, 40, 44 We have never detected ectopic chondrogenesis in the Gpr177 mutants. This difference might be attributed to the dual role of β-catenin in cell adhesion and signaling,29, 39, 40, 44, 48 because the loss of Gpr177 would not interfere with cell-cell interaction. It is possible that β-catenin–mediated cell adhesion regulates the lineage commitment of mesenchymal cells to chondrocytes. Recent analysis of mice with deficiency in only the transcriptional activation of β-catenin has unexpectedly revealed its function in cell adhesion, but not in Wnt signaling activation, which is critical for neural development.49 Using the transcription-deficient mutants of β-catenin permits dissecting its signaling and structural functions; further analysis may uncover new mechanisms underlying lineage commitment of the skeletal progenitors.
The differential phenotypes caused by the deletion of Gpr177 and β-catenin might also be attributed to the involvement of noncanonical Wnt in lineage commitment of mesenchymal cells. The noncanonical Wnt5a and Wnt5b are important regulators for chondrogenesis.50 Therefore, ectopic chondrogenesis detected in the β-catenin mutants might be caused by elevated signaling of noncanonical Wnt. Further investigation, focusing on the balance of canonical and noncanonical Wnt signaling, promises new insights into mesenchymal cell fate determination in skeletal development and disease.
All authors state that they have no conflicts of interest.
This work is supported by National Institutes of Health grant DE015654 to W.H. T.M. is a recipient of fellowship award from NYSTEM C026877. We thank Erestina Schipani and Matthew Hilton for reagents, Jiang Fu and Ansa Zahid for technical assistance, and Anthony Mirando for comments and suggestions.
Authors' roles: TM, MJ, and WH conceived, designed, and performed the experiments and analyzed the data. TM and WH wrote the paper.