The Wnt/β-Catenin Pathway Interacts Differentially with PTHrP Signaling to Control Chondrocyte Hypertrophy and Final Maturation

Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.


Introduction
Mammalian skeletal development is controlled by two mechanisms, endochondral and intramembranous bone formation. Endochondral ossification occurs in most parts of the body and requires a cartilage model prior to bone formation. Cartilage is composed of chondrocytes and in the developing long bones, these chondrocytes are organized into zones with distinct cellular morphologies and proliferation properties. Such organization is essential for the proper directional growth and elongation and is subject to tight regulation by secreted signaling molecules and transcription factors [1][2][3]. Proliferating chondrocytes are located at both ends of the cartilage and express Sox9 and Col2a1. Closer to the middle of the cartilage, proliferating chondrocytes become elongated and line up in columns along the longitudinal axis. These columnar chondrocytes divide quickly and begin to express Indian hedgehog (Ihh) and Runx2 as they exit the cell cycle to become prehypertrophic chondrocytes. Prehypertrophic chondrocytes then differentiate into hypertrophic chondrocytes that no longer express Sox9 and Col2a1, but express Col10a1 and angiogenic factors such as vascular endothelial growth factor (Vegf). Hypertrophic chondrocyes can be further divided into two populations according to their differences in gene expression and mineralization levels. Ihh and parathyroid hormone receptor 1 (Pthr1) are expressed only in prehypertrophic and early hypertrophic chondrocytes. The mature population of hypertrophic chondrocytes express Osteopontin (Opn) [4] and matrix metallo-proteinase 13 (Mmp13) that is required for invasion of blood vessels and osteoblasts to form the trabecular bone [5]. These most mature hypertrophic chondrocytes undergo apoptosis and are removed eventually. In addition, calcium deposition in the cartilage, which can be visualized by Von Kossa and Alizarin red staining, only occurs in the extracellular matrix of the final mature hypertrophic chondrocytes. Thus, both chondrocyte hypertrophy and final maturation are required for endochondral bone formation. However, it is not clear whether chondrocyte hypertrophy and final maturation are two consecutive developmental processes regulated by the same molecular pathways or they are actually developmentally separated and differentially regulated.
Ihh, parathyroid hormone related peptide (PTHrP), Wnts, Bone morphogenetic proteins (Bmps) and fibroblast growth factor (Fgfs) etc., regulate progressive chondrocyte proliferation and hypertrophy. PTHrP and Ihh are major regulators of chondrocyte hypertrophy by forming a negative feedback loop [6]. Ihh activates PTHrP expression and PTHrP then signals to proliferating chondrocytes to inhibit Ihh expression and chondrocyte hypertrophy by holding chondrocytes in a proliferating state. Wnt signaling pathways also regulate chondrocyte hypertrophy [7][8][9][10]. Among known Wnt signaling pathways, the canonical Wnt signaling pathway mediated by b-catenin [11] is the best understood and has been found to promote both chondrocyte hypertrophy and final maturation [9,10]. Wnt/b-catenin signaling acts independently of Ihh signaling to promote chondrocyte hypertrophy [12], but it is unknown whether Wnt/b-catenin and PTHrP signaling pathways regulate each other to control chondrocyte hypertrophy and maturation.
Here we have investigated the regulation of chondrocyte hypertrophy and final maturation and the genetic relationship between the canonical Wnt and PTHrP signaling pathways in sequential chondrocyte differentiation. We uncovered that chondrocyte hypertrophy and final maturation are two distinct processes that are differentially regulated by Wnt/b-catenin and PTHrP signaling. Canonical Wnt signaling promotes chondrocyte hypertrophy by antagonizing PTHrP signaling activity. However, the final maturation of hypertrophy chondrocytes is controlled by Wnt/b-catenin signaling independently of PTHrP signaling.

Wnt signaling controls chondrocyte hypertrophy by antagonizing PTHrP signaling
To test whether Wnt/b-catenin signaling acts through the PTHrP pathway in regulating chondrocyte hypertrophy, we first examined whether PTHrP expression was regulated by Wnt/bcatenin signaling in the developing long bone cartilage. b-catenin was removed in chondrocytes using the Col2a1-Cre mouse line [13]. The onset of chondrocyte hypertrophy at 12.5 days post coitum (dpc) were not altered [9]. However, continued initiation of chondrocyte hypertrophy by the remaining proliferating chondrocytes was slower in the Catnb c/c ;Col2a1-Cre mouse embryos [9]. PTHrP expression in the b-catenin deficient cartilage was similar to that in the control, but weaker at 14.5dpc (Fig. 1A). This is likely secondary to weakened Ihh expression in the cartilage of the Catnb c/c ; Col2a1-Cre mouse embryo [9,14], since ligand-independent activation of Hedgehog (Hh) signaling in the Catnb c/c ; Col2a1-Cre mice rescues PTHrP expression [12]. Furthermore, if Wnt/bcatenin signaling acts by regulating PTHrP expression to control chondrocyte hypertrophy, reduced PTHrP expression should lead to accelerated chondrocyte hypertrophy, the opposite of the observed phenotype for the Catnb c/c ; Col2a1-Cre mouse embryo [9,10]. These data suggest that Wnt/b-catenin signaling regulates chondrocyte hypertrophy independently of PTHrP expression.
The possibility remains that b-catenin interacts with PTHrP signaling in PTHrP-responding chondrocytes. To test this, we generated the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre double mutant mouse embryos. If Wnt/b-catenin signaling acts downstream of PTHrP signaling, initiation of chondrocyte hypertrophy should be similarly delayed in both the Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compared to that in the wild type embryo. Conversely, if Wnt/b-catenin signaling acts upstream of PTHrP signaling, initiation of chondrocyte hypertrophy should be similarly accelerated in the PTHrP 2/2 and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos. Single and double mutant embryos were harvested together with their wild type control littermates and analyzed by skeletal preparations. The relative length of Alizarin red stained segment (shows mineralized cells) versus Alcian blue stained segment (shows unmineralized chondrocytes) was compared in the same cartilage elements (Fig. 1B). Long bone mineralization was increased in the PTHrP 2/2 single mutant, but reduced in the Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos (Fig. 1B). These results suggest that final maturation of hypertrophic chondrocytes was similarly delayed in the Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre double mutant embryos.
Initiation and final maturation of chondrocyte hypertrophy were then examined more closely on histological sections of the limb at 14.5 dpc and 16.5 dpc. In both Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre double mutant embryos, Wnt signaling activity was similarly reduced as shown by the reduction in the expression of two transcriptional targets of canonical Wnt signaling, Lef1 and Tcf1 (Fig. 1C) [15,16]. Chondrocyte hypertrophy was then analyzed by Safranin O staining. Hypertrophic chondrocytes were those enlarged and lightly stained chondrocytes whereas proliferating chondrocytes were smaller and stained bright red. Surprisingly, although chondrocyte hypertrophy was delayed in the Catnb c/c ; Col2a1-Cre embryo, loss of PTHrP signaling leads to an acceleration of chondrocyte hypertrophy in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryo similar to that in the PTHrP 2/2 embryo (Fig. 1D). These morphological observations were further confirmed by molecular markers analysis. Col10a1 is expressed in hypertrophic chondrocytes whereas Col2a1 is expressed in proliferating chondrocytes. Col2a1 expression domain was reduced whereas Col10a1 expression domain was expanded to the same degree in the PTHrP 2/2 and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos at 14.5 dpc (Fig. 1D). Similar acceleration of chondrocyte hypertrophy in PTHrP 2/2 and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos was also observed in other skeletal elements such as the vertebral bodies (Fig. S1). These results indicate that Wnt/b-catenin signaling is dependent on PTHrP signaling in regulation of chondrocyte hypertrophy.
The above data suggest that active PTHrP signaling is required for Wnt/b-catenin signaling to regulate chondrocyte hypertrophy, but PTHrP signaling may not require b-catenin to regulate chondrocyte hypertrophy. This predicts that in the absence of bcatenin, PTHrP signaling levels can still determine the rate of chondrocyte hypertrophy. To test this, we need to partially reduce PTHrP signaling by reducing PTHrP expression. In this case, bcatenin removal should still enhance the residual PTHrP signaling, which will lead to a delay in chondrocyte hypertrophy. To achieve these genetic manipulations, we partially reduced  (Fig. 2B). In addition, we observed previously that when PTHrP expression was similarly upregulated in Ptch1 c/2 ; Col2a1-Cre and Ptch1 c/2 ; Catnb c/2 ; Col2a1-Cre embryos, there was a further delay of chondrocyte hypertrophy when b-catenin is removed [12]. Thus, in the absence of b-catenin, PTHrP signaling still inhibits chondrocyte hypertrophy in a dose-dependent manner, whereas in the absence of PTHrP signaling, b-catenin has no effect on chondrocyte hypertrophy.
Delayed chondrocyte hypertrophy can be secondary to increased proliferation [18,19]. Interestingly, chondrocyte proliferation was slightly increased in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compared to that in the PTHrP 2/2 single mutants at 16.5 dpc (Fig. S2), although chondrocyte hypertrophy was similar in both mutants. These results indicate that Wnt/b-catenin signaling can affect the rate at which cells divide in the proliferating chondrocyte pool, but only PTHrP signaling controls the brake that determines when chondrocytes exit cell cycle to undergo hypertrophy.

Wnt/b-catenin signaling is required for maturation of hypertrophic chondrocytes independently of PTHrP signaling
In the normal process of long bone development, chondrocyte hypertrophy is followed by hypertrophic chondrocyte maturation to allow the invasion of osteoblasts and blood vessels. Our observation that mineralization, but not hypertrophy, was similarly delayed in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre and Catnb c/c ; Col2a1-Cre embryos led us to think that formation of the early and late populations of hypertrophic chondrocytes are differentially regulated. To test this, we further examined chondrocyte maturation on histological sections. At 14.5 dpc, although Col10a1 expression domains were comparable between PTHrP 2/2 and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos (Fig. 1D), in stark contrast to PTHrP 2/2 embryo, where maturation of hypertrophic chondrocytes was also accelerated, hypertrophic chondrocyte maturation detected by von Kossa staining was significantly delayed in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre  (Fig. 3A). We then performed in situ hybridizations with probes specific to distinct stages of chondrocyte hypertrophy and maturation. At 14.5 dpc, expression domains of Mmp13 and Opn were much smaller in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compared to those in the PTHrP 2/2 embryos (Fig. 3A). By contrast, expression domains of Ihh and Pthr1, which mark prehypertrophic chondrocytes and early hypertrophic chondocytes, were expanded in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compared to that in the PTHrP 2/2 embryos (Fig. 3B). Thus, in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryo, loss of PTHrP signaling accelerated chondrocyte hypertrophy, but these hypertrophic chondrocytes were not able to undergo further maturation due to the blockage of Wnt/b-catenin signaling. As a result, the Ihh and Pthr1 expressing early hypertrophic chondrocyte population was significantly expanded. This increased Ihh expression domain in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryo likely caused increased osteoblast differentiation compared to that in the Catnb c/c ; Col2a1-Cre embryo (Fig. 3A and S3).
By 16.5 dpc, hypertrophic chondrocyte maturation, as indicated by von Kossa staining and expression of Mmp13 and Opn, caught up in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos with those in the PTHrP 2/2 embryos (Fig. 3C). However, at this stage more Col10a1-expressing hypertrophic chondrocytes were observed in the Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compare to the wild type control and the PTHrP 2/2 embryos, respectively (Fig. 3C). This is likely due to reduced elimination of mature hypertrophic chondrocytes caused by delayed blood vessel invasion and bone formation when b-catenin was removed [9]. Consistent with this, mineralized hypertrophic chondrocytes domains were increased in both Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compared to the wild type control and PTHrP 2/2 embryos, respectively (Fig. 3C). In addition, expression of Mmp9, which is a marker for osteoclasts and chondroclasts, was delayed in both Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos compared to the wild type control and PTHrP 2/2 embryos, respectively (Fig. 3E). However, Vegf expression, which was detected in hypertrophic chondrocytes was not decreased in Catnb c/c ; Col2a1-Cre and PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos (Fig. 3E). There was also no significant change in RANKL expression (Fig. S4). These results suggest that delayed blood vessel invasion and trabecular bone formation in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryo are at least in part a result of reduced expression of Mmps, not Vegf or RANKL. Interestingly, domains of Ihh/Pthr1 expressing-early hypertrophic chondrocytes were still expanded in the PTHrP 2/2 ; Catnb c/c ; Col2a1-Cre embryos at 16.5 dpc, particularly in the periphery of the cartilage compared to that in the PTHrP 2/2 embryos (Fig. 3D), which created a zone with a mixture of early and late mature hypertrophic chondrocytes. Taken together, these results indicate that the Wnt/b-catenin pathway regulates hypertrophic chondrocyte final maturation, at least partly and transiently, in a manner independent of PTHrP signaling.
Wnt signaling may regulate chondrocyte hypertrophy and maturation non-cell autonomously As PTHrP signaling controls chondrocyte hypertrophy cell autonomously [23], we tested whether the Wnt/b-catenin signaling pathway also acts cell autonomously. We expressed an activated form of b-catenin in subsets of chondrocytes using a mouse line with the exon 3 of b-catenin floxed (Catnb Ex3 ) [24]. This mouse line was crossed with a tamoxifen (TM) inducible Cre line, Col2a1-CreER [25]. As the tamoxifen induced Cre activity is not robust in all cells, the activated form of b-catenin is only expressed in a subset of chondrocytes in the Catnb Ex3/+ ; Col2a1-CreER embryos following TM injection. Since Lef1 is a transcription target of Wnt/b-catenin signaling [16], in these subset of chondrocytes, Lef1 expression was upregulated (Fig. 4A). In addition, bone formation was increased in the TM injected Catnb Ex3/+ ; Col2a1-CreER embryos indicated by von Kossa staining (Fig. 4A), suggesting that Wnt/ b-catenin signaling in chondrocytes may also promote bone formation in the perichondrium noncell autonomously. Interestingly, Safranin O staining and Col2a1 expression was significantly downregulated in the chondrocytes that expressed the activated form of b-catenin (Fig. 4A). However, these cells did not ectopically express Col10a1, a marker for hypertrophic chondrocytes (Fig. 4A). This is in sharp contrast to the normal process of chondrocyte hypertrophy in which Col2a1 expression is turned off, while Col10a1 expression is switched on. This is also drastically different from the Pthr1 2/2 clones in cartilage [23], in which loss of PTHrP signaling led to cell autonomous expression of Col10a1. In addition, while PTHrP signaling does not affect the determination of chondrocyte lineage [26,27], activated Wnt signaling in the developing cartilage can change chondrocyte identity to the ones that resemble cells found in the joint interzone or joint [28]. These data suggest that activated b-catenin signaling acts cell autonomously to control chondrocyte identity, but non-cell autonomously in regulating chondrocyte hypertrophy. Among the secreted signaling molecules that can regulate chondrocyte hypertrophy, Growth differentiation factor 5 (Gdf5) and Bmp2 expression are upregulated by Wnt/bcatenin signaling [28][29][30]. To address the potential role of these genes, we examined Bmp signaling activities in our mutant mice (Fig. 4B). In and around chondrocytes that express activated bcatenin, we observed stronger phosphorylation of Smad1, 5 and 8, indicating increased Bmp signaling [31]. As Bmp/Gdf5 signaling has been shown to promote chondrocyte hypertrophy [32][33][34], Gdf5 and Bmp2 are likely to participate in mediating the non-cell autonomous function of Wnt/b-catenin signaling in chondrocyte hypertrophy.

Discussion
We report here that during mouse embryonic cartilage development, Wnt/b-catenin signaling controls chondrocyte hypertrophy and final maturation by two distinct mechanisms. Wnt/b-catenin signaling regulates initiation of chondrocyte hypertrophy by antagonizing PTHrP signaling, whereas it acts independently of PTHrP signaling in controlling the final maturation of hypertrophic chondrocytes (Fig. 4C). Our results indicate that chondrocyte hypertrophy and final maturation are two separate developmental events that are regulated by distinct signaling interactions.
Our data suggests that delayed chondrocyte hypertrophy in the Catnb c/c ; Col2a1-Cre mutant embryo is due to upregulated PTHrP signaling, not enhanced PTHrP expression. As b-catenin is required for activating the expression of downstream targets of the canonical Wnt signaling pathway, a possible scenario is that one of the transcription targets of the canonical Wnt signaling pathway in the chondrocytes suppresses the PTHrP signaling activity. Alternatively, the b-catenin protein may directly suppress PTHrP signaling activity by interacting with PTHrP signaling components. Interaction of b-catenin with components of other signaling pathways may be an important mechanism underlying Wnt signaling cross regulation with other pathways. For instance, a LEF/TCF independent regulatory role of b-catenin in cell lineage determination has been demonstrated during pituitary gland development, in which b-catenin binds a specific homeodomain factor, Prop1, to activate expression of the critical lineagedetermining transcription factor, Pit1 [35]. However, if b-catenin inhibits PTHrP signaling by directly binding PTHrP signaling component(s), one may predict that PTHrP receptor and activated b-catenin both play a cell-autonomous role in chondrocyte hypertrophy. Although the PTHrP receptor acts cell autonomously in regulating chondrocyte hypertrophy [23], ectopic chondrocyte hypertrophy is not detected in chondrocytes expressing activated b-catenin as these cells have lost their chondrocyte identity because they express neither Col2a1 nor Col10a1. Interestingly, PTH activation of Pthr1 has been shown to activate the Wnt/b-catenin signaling directly by stabilizing b-catenin protein in osteoblasts [36]. However, in chondrocytes, it is unlikely that PTH/PTHrP signaling regulates chondrocyte hypertrophy through b-catenin as loss of PTH/PTHrP and Wnt/b-catenin signaling leads to opposite phenotypes.
Wnt/b-catenin signaling has two distinct roles in cartilage development. First, it inhibits chondrocyte cell lineage determination and maintenance [28]. Second, it promotes chondrocyte hypertrophy by inhibiting PTHrP signaling. However, such inhibition is likely to be incomplete. In addition, Wnt/b-catenin signaling may exert distinct functions in a dose dependent manner. It is conceivable that the two functions of Wnt/b-catenin signaling are also dose-dependent: Strong Wnt/b-catenin signaling (i.e., expression of activated b-catenin) inhibits chondrocyte cell fate determination and maintenance whereas weaker Wnt/b-catenin signaling promotes chondrocyte hypertrophy by reducing PTHrP signaling activities. Our results suggest that Wnt/b-catenin signaling may also interact with PTHrP signaling indirectly through a secondary signaling pathway. To this end, it will be interesting to further investigate genetically whether Gdf5/Bmp signaling control initiation chondrocyte hypertrophy by antagonizing PTHrP signaling and whether Gdf5/Bmp signaling also mediates the role of Wnt/b-catenin signaling in hypertrophic chondrocyte maturation.

Mice generation and Genotyping
Embryos were generated by crosses between mice carrying heterozygous PTHrP null allele PTHrP +/2 [26], b-catenin conditional allele Catnb c/c or gain-of-function allele Catnb ex3 , and a transgene Cre which is under the regulation of Col2a1 promoter and enhancer. Embryos were collected at 14.5, 15.5 and 16.5 dpc and embryos were analyzed as described. Genotyping for the Col2a1-Cre transgene and b-catenin alleles were performed as previously described [9].

Skeletal Preparation
Alizarin red and Alcian blue staining of skeletons was conducted as previously described [12]. Embryos were eviscated, fixed for 48 hours in ethanol and then transferred to acetone for 24 hours. Embryos were stained overnight at 37uC followed by 2 additional days at room temperature. Then embryos were cleared in 1% KOH and stored in 80% glycerol.

Tamoxifen preparation and injection
60 mg/ml Tamoxifen (Sigma) was dissolved in corn oil (Sigma) and sonicated until the solution became clear. The solution was filtered and 0.05 ml was injected intraperitoneally into the pregnant female mice at 13.5 dpc and the embryos were harvested at 17.5 dpc.