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.

Human craniosynostosis syndromes, resulting from activating or neomorphic mutations in fibroblast growth factor receptor 2 (FGFR2), underscore an essential role for FGFR2 signaling in skeletal development. Embryos harboring homozygous null mutations in FGFR2 die prior to skeletogenesis. To address the role of FGFR2 in normal bone development, a conditional gene deletion approach was adopted. Homologous introduction of cre recombinase into the Dermo1 (Twist2) gene locus resulted in robust expression of CRE in mesenchymal condensations giving rise to both osteoblast and chondrocyte lineages.Inactivation of a floxed Fgfr2 allele with Dermo1-cre resulted in mice with skeletal dwarfism and decreased bone density. Although differentiation of the osteoblast lineage was not disturbed, the proliferation of osteoprogenitors and the anabolic function of mature osteoblasts were severely affected.

Signals that govern development of the osteoblast lineage are not well understood. Indian hedgehog (Ihh), a member of the hedgehog (Hh) family of proteins, is essential for osteogenesis in the endochondral skeleton during embryogenesis. The canonical pathway of Wnt signaling has been implicated by studies of Lrp5, a co-receptor for Wnt proteins, in postnatal bone mass homeostasis. In the present study we demonstrate that β-catenin, a central player in the canonical Wnt pathway, is indispensable for osteoblast differentiation in the mouse embryo. Moreover, we present evidence that Wnt signaling functions downstream of Ihh in development of the osteoblast lineage. Finally Wnt7b is identified as a potential endogenous ligand regulating osteogenesis. These data support a model that integrates Hh and Wnt signaling in the regulation of osteoblast development.

The epicardium regulates growth and survival of the underlying myocardium. This activity depends on intrinsic retinoic acid (RA) and erythropoietin signals. However, these signals do not act directly on the myocardium and instead are proposed to regulate the production of an unidentified soluble epicardial derived mitogen. Here, we show that Fgf9, Fgf16, and Fgf20 are expressed in the endocardium and epicardium and that RA can induce epicardial expression of Fgf9. Using knockout mice and an embryonic heart organ culture system, we show that endocardial and epicardial derived FGF signals regulate myocardial proliferation during midgestation heart development. We further show that this FGF signal is received by both FGF receptors 1 and 2 acting redundantly in the cardiomyoblast. In the absence of this signal, premature differentiation results in cellular hypertrophy and newborn mice develop a dilated cardiomyopathy. FGFs thus constitute all or part of the epicardial signal regulating myocardial growth and differentiation.

SummaryAbnormalities in cartilage and bone development in the Apert syndrome FGFR2 +/S252W