Expression and function of Dlx genes in the osteoblast lineage

AbstractOur laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5−/−/Dlx6−/− mice have more severe craniofacial and limb defects than Dlx5−/−, some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5−/− mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function

FGF/FGFR3 signaling and fate of avian Meckel\u27s cartilage

The midportion of avian Meckel\u27s cartilage (MC) is a permanent cartilage that persists into adult life. The overall goal of our studies is to gain insight into the genetic pathway(s) involved in preventing the maturation/hypertrophy in c&barbelow;hondrocytes in the m&barbelow;idportion of M&barbelow;eckel\u27s c&barbelow;artilage (CMMC) in vivo. First, we determined the stage of differentiation of CMMC. We showed that CMMC maintained high levels of expression of aggrecan, sox9, and Fgfr3 but lacked the expression of type-X-collagen, Ihh, Cbfa1, BmpIA, and Fgfr1 . Low but detectable levels of PTH1R and BmprIB were also expressed in CMMC. PTHrP and Fgfr2 were expressed in the perichondrium. Thus, CMMC are maintained/arrested in the late proliferative stage of differentiation and do not enter the prehypertrophic stage of maturation. The sustained expression of Fgfr3 and PTH1R in CMMC suggest that the inability of CMMC to progress into the prehypertrophic stage of maturation is related to the activities of these negative signaling pathways. Our observations also indicate that despite their inability to mature in vivo, CMMC have the ability to mature and express hypertrophy-associated traits in vitro. Both FGF2 (a ligand for FGFR3) and PTHrP modulated the in vitro-induced maturation and hypertrophy of CMMC. ^ We also generated retroviral constructs expressing epitope tagged, dominant-negative forms of murine FGFR3 (RCAS-dn-mFGFR3). Overexpression of RCAS-dn-mFGFR3 in vivo caused numerous abnormalities in mandibular development on the injected side in a stage-specific manner. At early stages of development RCAS-dn-mFGFR3 caused decreases in the length of mandibular processes and Meckel\u27s cartilage, partial or complete absence of mandibular bones. On the other hand, this treatment at later stages of development did not affect the outgrowth of the mandibular process and MC but resulted in localized thickening of MC, increases in the length of MC and partial to complete absence of most mandibular membranous bones. Our results provide evidence for involvement of FGFR3 signaling in several aspects of chick mandibular morphogenesis.