Wnt signaling via the Frizzled (Fz) receptor controls cell polarity and movement during development, but the molecular nature of Wnt/Fz polarity signal transduction remains poorly defined. Here we report that in human cells and during Xenopus embryogenesis, Wnt/Fz signaling activates the small GTPase Rho, a key regulator of cytoskeleton architecture. Wnt/Fz activation of Rho requires the cytoplasmic protein Dishevelled (Dvl) and a novel Formin homology protein Daam1. Daam1 binds to both Dvl and Rho, and mediates Wnt-induced Dvl-Rho complex formation. Inhibition or depletion of Daam1 prevents Wnt/Fz activation of Rho and of Xenopus gastrulation, but not of beta-catenin signaling. Our study illustrates a molecular pathway from Wnt/Fz signaling to Rho activation in cell polarity signal transduction.

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline-phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.

Regulation of ␤-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of ␤-catenin by the Axin-glycogen synthase kinase (GSK)-3␤ complex targets ␤-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize ␤-catenin and cause human cancers. How ␤-catenin phosphorylation results in its degradation is undefined. Here we show that phosphorylated ␤-catenin is specifically recognized by ␤-Trcp, an F-box͞WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. ␤-catenin harboring mutations at the critical phosphoserine residues escapes recognition by ␤-Trcp, thus providing a molecular explanation for why these mutations cause ␤-catenin accumulation that leads to cancer. Inhibition of endogenous ␤-Trcp function by a dominant negative mutant stabilizes ␤-catenin, activates Wnt͞␤-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, ␤-Trcp plays a central role in recruiting phosphorylated ␤-catenin for degradation and in dorsoventral patterning of the Xenopus embryo.