Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.

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Chondrocyte differentiation is a multi-step process characterized by successive changes in cell morphology and gene expression. In addition to tight regulation by numerous soluble factors, these processes are controlled by adhesive events. During the early phase of the chondrocyte life cycle, cell-cell adhesion through molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) is required for differentiation of mesenchymal precursor cells to chondrocytes. At later stages, for example in growth plate chondrocytes, adhesion signaling from extracellular matrix (ECM) proteins through integrins and other ECM receptors such as the discoidin domain receptor (DDR) 2 (a collagen receptor) and Annexin V is necessary for normal chondrocyte proliferation and hypertrophy. Cell-matrix interactions are also important for chondrogenesis, for example through the activity of CD44, a receptor for Hyaluronan and collagens. The roles of several signaling molecules involved in adhesive signaling, such as integrin-linked kinase (ILK) and Rho GTPases, during chondrocyte differentiation are beginning to be understood, and the actin cytoskeleton has been identified as a common target of these adhesive pathways. Complete elucidation of the pathways connecting adhesion receptors to downstream effectors and the mechanisms integrating adhesion signaling with growth factor-and hormone-induced pathways is required for a better understanding of physiological and pathological skeletal development.

The development of the cartilage template that precedes endochondral bone formation requires the condensation of mesenchymal cells and their subsequent differentiation to the chondrocytic lineage. We have previously shown that inhibition of the RhoA/ ROCK signaling pathway or actin dynamics enhances Sox9 mRNA expression, increases glycosaminoglycan production, and transforms cell shape to a spherical, chondrocyte-like morphology. However, we demonstrate here that in three-dimensional micromass cultures of mesenchymal cells, increased expression of Sox9 in response to these manipulations is not sufficient to induce the expression of established Sox9 target genes. This is illustrated by a decrease in the transcript levels of collagen II and aggrecan as well as reduced activity of a Sox9-responsive reporter gene in response to ROCK inhibition and cytochalasin D. We also demonstrate a decrease in mRNA levels of the transcriptional co-activators L-Sox5 and Sox6 upon ROCK inhibition and cytochalasin D. The decrease in Sox9 activity is likely partially due to reduced L-Sox5 and Sox6 levels but also to a delay in Sox9 phosphorylation following ROCK inhibition. In contrast, inhibition of the RhoA/ROCK pathway and cytochalasin D treatment in monolayer culture results in the enhancement of a number of markers of chondrogenesis such as Sox9 activity and collagen II and aggrecan transcripts levels. These data demonstrate that the effects of RhoA/ROCK signaling and actin polymerization inhibitors on chondrogenic gene expression are dependent on the cellular context.Endochondral ossification is a developmental process that forms the majority of the mammalian skeleton, as reviewed in Ref. 1. Beginning with condensations of mesenchymal cells and subsequent differentiation to the chondrocytic lineage, precisely shaped cartilage templates are formed (2). These templates are composed of proliferating and differentiating chondrocytes as well as vast amounts of extracellular matrix produced by chondrocytes (3). The commitment of cells to the chondrocytic lineage is marked by change in cell shape (from a spread, fibroblastic-like morphology to a spherical morphology), expression of the essential transcription factor Sox9 (4) and the matrix markers collagen II (5) and aggrecan (6), and increased glycosaminoglycan production (1).Although the importance of cell shape is recognized (7), molecular mechanisms controlling these processes had not been studied extensively.The RhoA/ROCK signaling pathway is an excellent candidate as its role as a master regulator of the actin cytoskeleton has been well documented (8 -10). In addition, RhoA signaling controls cell cycle progression (11), differentiation (12), and apoptosis in other cell types (13). We recently demonstrated that inhibition of the RhoA/ROCK pathway by the pharmacological inhibitor Y27632 induced spherical cell morphology, increased the transcription of the chondrogenic transcription factor Sox9, and enhanced glycosaminoglycan accumulation (14). We also demonstrated that RhoA overe...

Crosstalk, also known as ghosting or leakage, is a primary factor in determining the image quality of stereoscopic three dimensional (3D) displays. In a stereoscopic display, a separate perspective view is presented to each of the observer's two eyes in order to experience a 3D image with depth sensation. When crosstalk is present in a stereoscopic display, each eye will see a combination of the image intended for that eye, and some of the image intended for the other eye-making the image look doubled or ghosted. High levels of crosstalk can make stereoscopic images hard to fuse and lack fidelity, so it is important to achieve low levels of crosstalk in the development of high-quality stereoscopic displays. Descriptive and mathematical definitions of these terms are formalized and summarized. The mechanisms by which crosstalk occurs in different stereoscopic display technologies are also reviewed, including micropol 3D liquid crystal displays (LCDs), autostereoscopic (lenticular and parallax barrier), polarized projection, anaglyph, and time-sequential 3D on LCDs, plasma display panels and cathode ray tubes. Crosstalk reduction and crosstalk cancellation are also discussed along with methods of measuring and simulating crosstalk.