Cartilage-specific ß-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development.

The WNT/ß-catenin signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. Although the importance of ß-catenin signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate ß-catenin in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for ß-catenin in committed growth-plate chondrocytes has been less robust. To identify the specific role of cartilage-derived ß-catenin in regulating cartilage and bone development, we studied chondrocyte-specific gain- and loss-of-function genetic mouse models using the tamoxifen-inducible Col2Cre(ERT2) transgene in combination with ß-catenin(fx(exon3)/wt) or ß-catenin(fx/fx) floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage-specific ß-catenin signaling promotes chondrocyte maturation, possibly involving a bone morphogenic protein 2 (BMP2)-mediated mechanism. Second, cartilage-specific ß-catenin facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced matrix metalloproteinase (MMP) expression at sites of cartilage degradation, and potentially by enhancing Indian hedgehog (IHH) signaling activity to recruit vascular tissues. Finally, cartilage-specific ß-catenin signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage-derived ß-catenin signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation.

Axin2 regulates chondrocyte maturation and axial skeletal development.

Axis inhibition proteins 1 and 2 (Axin1 and Axin2) are scaffolding proteins that modulate at least two signaling pathways that are crucial in skeletogenesis: the Wnt/beta-catenin and TGF-beta signaling pathways. To determine whether Axin2 is important in skeletogenesis, we examined the skeletal phenotype of Axin2-null mice in a wild-type or Axin1(+/-) background. Animals with disrupted Axin2 expression displayed a runt phenotype when compared to heterozygous littermates. Whole-mount and tissue beta-galactosidase staining of Axin2(LacZ/LacZ) mice revealed that Axin2 is expressed in cartilage tissue, and histological sections from knockout animals showed shorter hypertrophic zones in the growth plate. Primary chondrocytes were isolated from Axin2-null and wild-type mice, cultured, and assayed for type X collagen gene expression. While type II collagen levels were depressed in cells from Axin2-deficient animals, type X collagen gene expression was enhanced. There was no difference in BrdU incorporation between null and heterozygous mice, suggesting that loss of Axin2 does not alter chondrocyte proliferation. Taken together, these findings reveal that disruption of Axin2 expression results in accelerated chondrocyte maturation. In the presence of a heterozygous deficiency of Axin1, Axin2 was also shown to play a critical role in craniofacial and axial skeleton development.

Axin1 and Axin2 are regulated by TGF- and mediate cross-talk between TGF- and Wnt signaling pathways.

Chondrocyte maturation during endochondral bone formation is regulated by a number of signals that either promote or inhibit maturation. Among these, two well-studied signaling pathways play crucial roles in modulating chondrocyte maturation: transforming growth factor-beta (TGF-beta)/Smad3 signaling slows the rate of chondrocyte maturation, while Wingless/INT-1-related (Wnt)/beta-catenin signaling enhances the rate of chondrocyte maturation. Axin1 and Axin2 are functionally equivalent and have been shown to inhibit Wnt/beta-catenin signaling and stimulate TGF-beta signaling. Here we show that while Wnt3a stimulates Axin2 in a negative feedback loop, TGF-beta suppresses the expression of both Axin1 and Axin2 and stimulates beta-catenin signaling. In Axin2 -/- chondrocytes, TGF-beta treatment results in a sustained increase in beta-catenin levels compared to wild-type chondrocytes. In contrast, overexpression of Axin enhanced TGF-beta signaling while overexpression of beta-catenin inhibited the ability of TGF-beta to induce Smad3-sensitive reporters. Finally, the suppression of the Axins is Smad3-dependent since the effect is absent in Smad3 -/- chondrocytes. Altogether these findings show that the Axins act to integrate signals between the Wnt/beta-catenin and TGF-beta/Smad pathways. Since the suppression Axin1 and Axin2 expression by TGF-beta reduces TGF-beta signaling and enhances Wnt/beta-catenin signaling, the overall effect is a shift from TGF-beta toward Wnt/beta-catenin signaling and an acceleration of chondrocyte maturation.

Adaptation to sine-wave gratings selectively reduces the contrast gain of the adapted stimuli.

Adapting to sinusoidal gratings selectively reduces contrast sensitivity to subsequent test stimuli. To investigate the perceptual processes underlying selective adaptation, we developed an external noise plus adaptation paradigm and a theoretical framework based on a noisy observer model (the contrast-gain-control Perceptual Template Model [cgcPTM]). After adapting to a 45 deg, 2-Hz counter-flickering sine grating of 0.8 contrast, observers performed two-interval forced-choice detection of Gabors of matched spatial frequency, tilted at either 45 or 135 deg and embedded in one of six levels of white external noise (Experiment 1) or embedded in orientation band-pass-filtered external noise (Experiment 2). On the basis of the cgcPTM, we found that adaptation selectively reduced the contrast gain of the perceptual template at the adapted spatial frequency and orientation without altering either pre- or post-gain-control (additive and multiplicative) noises or changing transducer nonlinearity. Modeled as notches on the perceptual templates, the estimated full orientation bandwidth of adaptation at half height was about 8.3 deg.