A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. To elucidate the function of Cbfa1, we generated mice with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 died just after birth without breathing. Examination of their skeletal systems showed a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in the cartilage. Therefore, our data suggest that both intramembranous and endochondral ossification were completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice, and demonstrate that Cbfa1 plays an essential role in osteogenesis.

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Transforming growth factor ␤ (TGF-␤) is a potent multifunctional regulator of cell growth and differentiation. Although nearly all cells synthesize and respond to TGF-␤, bone and cartilage are particularly rich in this growth factor (6, 46). TGF-␤1, the prototypic member of the TGF-␤ superfamily, elicits diverse cellular responses, including (i) inhibition of adipogenesis and myogenesis and (ii) stimulation of chondrogenesis and osteogenesis (31). TGF-␤1 stimulates the synthesis of matrix proteins and their receptors (for example, fibronectin, fibronectin receptor, collagen, osteonectin, osteopontin, and integrins) and inhibits matrix degradation by increasing the production of protease inhibitors and decreasing the production of proteases (42). Members of the TGF-␤ superfamily with important effects on bone cell differentiation are bone morphogenetic proteins (BMPs) (17, 41), which were first identified as factors that induce bone formation in vivo when implanted into muscular tissues (54). Unlike TGF-␤, which induces new bone formation only when injected near bone, BMPs produce bone formation even when injected into ectopic sites. TGF-␤ and BMPs bind to distinct receptors, TGF-␤ type I and II receptors for TGF-␤ and BMP type I and II receptors for BMPs. Following ligand binding, the receptor-associated kinase is activated and phosphorylates Smads, which move into the nucleus to stimulate the transcription of a set of target genes. Smad2 and -3 are activated by TGF-␤ receptors and mediate TGF-␤ responses, whereas Smad1, -5, and -8 are activated by BMP receptors and transduce BMP signals (15,32,57).The pluripotent mesenchymal precursor cell line C2C12 provides a model system to study the early stage of osteoblast differentiation during bone formation in muscular tissues. In this model, TGF-␤1 inhibits the differentiation of C2C12 cells into multinucleated myotubes without inducing osteoblast phenotypes. BMP-2 not only inhibits the terminal differentiation of C2C12 cells but also induces osteoblast phenotypes (20). Therefore, the C2C12 model is useful for analyzing both the common and specific signaling mechanisms of TGF-␤ and BMPs. In C2C12 cells, overexpression of Smad1 and Smad5 induced alkaline phosphatase (ALP) activity, a typical osteoblast-specific marker, and inhibited muscle-specific gene expression (11,36,56). These results suggested that BMP functions via either Smad1 or Smad5 and that the induction of the osteoblast phenotype and the inhibition of myogenic differentiation are regulated at the transcriptional level. However, the molecular mechanisms through which Smads block myogenic differentiation and induce osteogenic differentiation are not known.Runx/PEBP2/Cbf (hereafter referred to as Runx) is a sequence-specific DNA binding protein that recognizes a specific DNA sequence originally identified as the binding site for

Runx2, osterix, and b-catenin are essential for osteoblast differentiation. Runx2 directs multipotent mesenchymal cells to an osteoblastic lineage, and inhibits them from differentiating into the adipocytic and chondrocytic lineages. After differentiating to preosteoblasts, b-catenin, osterix, and Runx2 direct them to immature osteoblasts, which produce bone matrix proteins, blocking their potential to differentiate into the chondrocytic lineage. Runx2 inhibits osteoblast maturation and the transition into osteocytes, keeping osteoblasts in an immature stage. Other transcription factors including Msx1, Msx2, Dlx5, Dlx6, Twist, AP1(Fos/Jun), Knox-20, Sp3, and ATF4 are also involved in osteoblast differentiation. To gain an understanding of bone development, it is important to position these transcription factors to the right places in the processes of osteoblast differentiation.

The differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed inVertebrate skeletons are constructed through the formation of bone structures, a process that is achieved by intramembranous or endochondral ossification. Intramembranous bones, which are directly formed by osteoblasts, are restricted to the cranial vault, some facial bones, and parts of the mandible and clavicle, whereas the rest of the skeleton is composed of endochondral bones that are formed as a cartilaginous template which is then replaced by bone. In early skeletal development, mesenchymal cells condense and acquire the phenotypes of chondrocytes including the ability to produce Col2a1 and proteoglycan. In the process of endochondral ossification, immature chondrocytes proliferate, and chondrocytes at the center of the cartilaginous skeleton begin to mature to become prehypertrophic chondrocytes, which express parathyroid hormone/parathyroid hormone-related peptide (Pthlh) receptor (Pthr1) and Indian hedgehog (Ihh). The prehypertrophic chondrocytes further mature to hypertrophic chondrocytes, which express Col10a1. Upon the terminal differentiation of chondrocytes, the terminal hypertrophic chondrocytes express osteopontin, the matrix is mineralized, vascular vessels invade the calcified cartilage, and finally the cartilage is replaced by bone. Chondrocyte proliferation and differentiation occur in an organized manner and result in the formation of a growth plate that is composed of layers of chondrocytes at different stages of differentiation, in-