Title

Functional studies of Dlx5 during bone formation: Implications of Dlx genes in promoting osteoblast differentiation

Date of Completion

January 2007

Keywords

Biology, Molecular|Biology, Genetics|Biology, Cell

Degree

Ph.D.

Abstract

Genetic approaches have been undertaken to investigate how overexpression or loss of Dlx5 expression alters osteogenesis. When Dlx5 is overexpressed in mouse primary osteoblast cultures via viral mediated transduction, osteoblast differentiation is more robust. However, only minimal defects in osteoblast differentiation were observed in primary osteoblast cultures from Dlx5 knock out mice. Thorough histomorphometry analysis of long bones from Dlx5 knock out mice did not show evidence that Dlx5 has a significant role in the long bone formation. This may be due to a functional redundancy that exists between Dlx5 and other members of the Dlx family. To provide further support for redundant roles between different Dlx family members during bone formation, the expression patterns of all members of Dlx gene family during the osteoblast maturation process was characterized by real time PCR, which clearly implicated Dlx2, Dlx3 and Dlx6 as other potentially important regulators of osteogenesis. Moreover, studies using a dominant negative Dlx retroviral virus expression vector strongly impaired osteogenesis suggesting that the Dlx gene family is indispensable for osteoblast differentiation. Finally, we utilized Dlx5 over expression mouse models to further explore Dlx5 function in bone formation in vivo. Two separate groups of Dlx5 over expressing mice were generated. One group over expressed Dlx5 at the early stage of osteoblast lineage using 3.6 kb rat Collal promoter (3.6Dlx5), the other group over expressed Dlx5 in mature osteoblasts and osteocytes using 1.7 kb rat Collal promoter (1.7Dlx5). We observed endogenous Dlx genes have this temporal expression patterns during osteoblast differentiation, therefore, study transgenic mice overexpressing Dlx5 in the early vs. late stage of osteoblast lineage will provide us comprehensive information to understand the role of Dlx gene family in regulating skeletal development. Our results indicated that 1.7Dlx5 transgenic mice have increased bone formation, whereas 3.6Dlx5 mice have decreased bone volume. Both transgenic lines showed increased osteoblast activation and function. Thorough evaluation of these transgenic animals indicated that osteoprogenitor recruitment and (or) expansion may be responsible for the osteopenia in 3.6Dlx5 mice. We demonstrated that gain of function studies are more appropriate models to understand Dlx genes in regulating bone formation.^