One of the major roles of chondrocytes during endochondral bone formation is the paracrine secretion of Ihh that is responsible for the specification of endochondral osteoprogenitors and the initiation of Runx2 expression (St-Jacques

INTRODUCTION The mammalian skull vault, which protects the brain, comprises bones derived from dual embryonic origins. Cells from both cranial neural crest (CNC) and paraxial mesoderm (PM) form skull progenitors, and these distinct populations are already spatially segregated in rostral and caudal domains above the eye in the mouse at E11.5 (Yoshida et al., 2008). Skull bones undergo intramembranous ossification, in which osteoprogenitors are specified and subsequently differentiate into lineage-restricted osteoblasts, moving dorsally before ossifying directly as bone (Yoshida et al., 2008). In the mouse, CNC contributes to the frontal bones and the interparietal bone, and PM gives rise to the parietal bones of the posterior skull vault (Jiang et al., 2002; Yoshida et al., 2008). Bone formation occurs through two divergent mechanisms: intramembranous and endochondral ossification. The majority of the skeleton undergoes endochondral bone formation, in which a cartilage template first forms, becomes mineralized, and is replaced by osteoblast-made bone. By contrast, intramembranous bones, such as those of the skull, form by direct ossification without a cartilage intermediate. Bone-forming osteoblasts of both intramembranous and endochondral bones express the transcription factors Runx2 and Osterix (Osx; Sp7 – Mouse Genome Informatics), which are essential for initial osteoprogenitor specification and osteoblast lineage commitment, respectively (Nakashima et al., 2002; Otto et al., 1997). In these early stages of progenitor formation in endochondral bones, genetic studies have demonstrated that the BMP, Wnt, Indian hedgehog (Ihh) and Notch signaling pathways are important; however, a direct examination of their roles during the development of intramembranous bone progenitors is lacking (reviewed by Long, 2008). Several studies have revealed differences in the molecular signals required for the specification of endochondral and intramembranous bone progenitors. For example, the chondrogenic determinant Sox9 is essential for the formation of the cartilage anlage, which precedes endochondral progenitor specification. Consequently, Sox9 mutant cells from chimaeric embryos do not contribute to endochondral bone (Bi et al., 1999). One of the major roles of chondrocytes during endochondral bone formation is the paracrine secretion of Ihh that is responsible for the specification of endochondral osteoprogenitors and the initiation of Runx2 expression (St-Jacques et al., 1999). By contrast, Sox9 and chondrogenesis are dispensable in skull vault morphogenesis, consistent with the finding that intramembranous bone progenitors do not require Ihh for intramembranous osteoprogenitor specification (St-Jacques et al., 1999). Therefore, molecular differences in the initiation of osteoprogenitor cell specification between these two ossification programs are evident, and yet the signaling pathways that specify intramembranous bone progenitors remain unidentified. Since skull vault formation occurs in the absence of an intermediary cartilage, it is then plausible that the pathway initiating osteoprogenitor specification in this population might simultaneously inhibit chondrocyte formation in the skull. Several studies have confirmed that osteoblasts and chondrocytes involved in endochondral bone formation originate from common progenitors (Akiyama et al., 2005). -catenindependent signaling is crucial in determining the definitive cell fate, and it inhibits chondrocyte differentiation in endochondral osteoprogenitors (Hill et al., 2005). -catenin transduces Wnt signaling through the Frizzled and LRP receptors by associating with members of the TCF/LEF transcription factor family in the nucleus and regulating target gene expression (Bhanot et al., 1996; Korinek et al., 1998; Liu et al., 1999; Tamai et al., 2000). -catenin is also required for intramembranous skull vault mineralization (Day et al., 2005; Hill et al., 2005). Conditional -catenin deletion 1Department of Pathology, 3Department of Biology, 5Department of Genetics and 6Department of Dermatology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. 2Department of Pharmacology and 4Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA.