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Runx2 is necessary and sufficient for osteoblast differentiation, yet its expression precedes the appearance of osteoblasts by 4 days. Here we show that Twist proteins transiently inhibit Runx2 function during skeletogenesis. Twist-1 and -2 are expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurs only after their expression decreases. Double heterozygotes for Twist-1 and Runx2 deletion have none of the skull abnormalities observed in Runx2(+/-) mice, a Twist-2 null background rescues the clavicle phenotype of Runx2(+/-) mice, and Twist-1 or -2 deficiency leads to premature osteoblast differentiation. Furthermore, Twist-1 overexpression inhibits osteoblast differentiation without affecting Runx2 expression. Twist proteins' antiosteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runx2 DNA binding domain to inhibit its function. In vivo mutagenesis confirms the antiosteogenic function of the Twist box. Thus, relief of inhibition by Twist proteins is a mandatory event precluding osteoblast differentiation.

We describe a novel transgene insertional mouse mutant with skeletal abnormalities characterized by a kinked tail and severe curvature of the spine. The disrupted locus is designated kkt for "kyphoscoliosis kinked tail." Malformed vertebrae including bilateral ossification centers and premature fusion of the vertebral body to the pedicles are observed along the vertebral column, and the lower thoracic and lumbar vertebrae are the most affected. Some of the homozygous kkt neonates displayed two backward-pointing transverse processes in the sixth lumbar vertebra (L6) that resembled the first sacral vertebra, and some displayed one forward- and one backward-pointing transverse process in L6. The fourth and fifth sternebrae were also fused, and the acromion process of the scapula was missing in kkt mice. The skeletal abnormalities are similar to those observed in the mouse mutant undulated (un). The transgene is integrated at the distal end of chromosome 2 close to the Pax1 gene, as revealed by FISH analysis. However, mutation of the Pax1 gene is responsible for the un phenotype, but the Pax1 gene in the kkt mice is not rearranged or deleted. Pax1 is expressed normally in kkt embryos and in the thymus of mature animals, and there is no mutation in its coding sequence. Thus, the skeletal abnormalities observed in the kkt mutant are not due to a lack of functional Pax1. Mouse genomic sequences flanking the transgene and PAC clones spanning the wild-type kkt locus have been isolated, and reverse Northern analysis showed that the PACs contain transcribed sequence. Compound heterozygotes between un and kkt (un(+/-)/kkt(+/-)) display skeletal abnormalities similar to those of un or kkt homozygotes, but they have multiple lumbar vertebrae with a split vertebral body that is more severe than in homozygous un or kkt neonates. Furthermore, the sternebrae are not fused and no backward-pointing transverse processes are detected in L6. It is therefore apparent that these two mutations do not fully complement each other, and we propose that a gene in the kkt locus possesses a unique role that functions in concert with Pax1 during skeletal development., (Copyright 2000 Academic Press.)