In vivo impact of Dlx3 conditional inactivation in neural crest‐derived craniofacial bones

Abstract

Mutations in DLX3 in humans lead to defects in craniofacial and appendicular bones, yet the in vivo activity related to Dlx3 function during normal skeletal development have not been fully elucidated. Here we used a conditional knockout approach to analyze the effects of neural crest deletion of Dlx3 on craniofacial bones development. At birth, mutant mice exhibit a normal overall positioning of the skull bones, but a change in the shape of the calvaria was observed. Molecular analysis of the genes affected in the frontal bones and mandibles from these mice identified several bone markers known to affect bone development, with a strong prediction for increased bone formation and mineralization in vivo. Interestingly, while a subset of these genes were similarly affected in frontal bones and mandibles (Sost, Mepe, Bglap, Alp, Ibsp, Agt), several genes, including Lect1 and Calca, were specifically affected in frontal bones. Consistent with these molecular alterations, cells isolated from the frontal bone of mutant mice exhibited increased differentiation and mineralization capacities ex vivo, supporting cell autonomous defects in neural crest cells. However, adult mutant animals exhibited decreased bone mineral density in both mandibles and calvaria, as well as a significant increase in bone porosity. Together, these observations suggest that mature osteoblasts in the adult respond to signals that regulate adult bone mass and remodeling. This study provides new downstream targets for Dlx3 in craniofacial bone, and gives additional evidence of the complex regulation of bone formation and homeostasis in the adult skeleton.

DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo

“…Similar to the present study, Wnt1cre:Dlx3 mice exhibited decreased BMD in mandibular and calvarial bones and increased porosity in mandible. 12 Furthermore, here we show that adenoviral-Cre-infected calvarial cells and longbone BMSCs isolated from Dlx3 F/F neonates and 5 wk mice, respectively, showed increased differentiation capacity as demonstrated by ALPL staining, which is consistent with calvarial cell differentiation from Wnt1cre:Dlx3 neonates. 12 However, craniofacial bones from Wnt1cre:Dlx3 neonates and femurs from Dlx3 OCN-cKO 5 wk mice had a particular gene signature where few genes including Alpl, Ibsp, Mepe, Ihh and Adamst18 were commonly affected.…”

“…12 Furthermore, here we show that adenoviral-Cre-infected calvarial cells and longbone BMSCs isolated from Dlx3 F/F neonates and 5 wk mice, respectively, showed increased differentiation capacity as demonstrated by ALPL staining, which is consistent with calvarial cell differentiation from Wnt1cre:Dlx3 neonates. 12 However, craniofacial bones from Wnt1cre:Dlx3 neonates and femurs from Dlx3 OCN-cKO 5 wk mice had a particular gene signature where few genes including Alpl, Ibsp, Mepe, Ihh and Adamst18 were commonly affected. We presently show that although expression of Runx2 and Dlx5 were significantly upregulated in Dlx3-deleted BMSCs from long bones of 5 wk Dlx3 F/F , these genes were not affected in Dlx3-deleted calvarial cells isolated from Dlx3 F/F neonates.…”

“…This was further supported by ex vivo assays on frontal bone osteoblasts, suggesting an inhibitory role for DLX3 in osteoblastic differentiation. 12 Contrary to this prediction, adult mice exhibited decreased BMD and increased porosity in neural crest-derived craniofacial bones. 12 A transgenic mouse model expressing the TDO DLX3 gene mutation driven by the osteoblast-specific 2.3 Col1A1 promoter was characterized by Choi et al 13 This model resulted in increased trabecular bone volume in young and adult mice; however, the diaphysis phenotype was not described.…”

“…9 Recently, we investigated the effects of neural crest deletion of Dlx3 in craniofacial bones. 12 The gene signature of craniofacial bones from Wnt1cre:Dlx3 neonates predicted increased bone formation and mineralization. This was further supported by ex vivo assays on frontal bone osteoblasts, suggesting an inhibitory role for DLX3 in osteoblastic differentiation.…”

“…12 A transgenic mouse model expressing the TDO DLX3 gene mutation driven by the osteoblast-specific 2.3 Col1A1 promoter was characterized by Choi et al 13 This model resulted in increased trabecular bone volume in young and adult mice; however, the diaphysis phenotype was not described. Despite bone marrow stromal cells (BMSCs) from mutants exhibiting enhanced osteoblastic differentiation and increased bone marker expression ex vivo, the dynamic bone formation rate was not increased in vivo and the trabecular phenotype was attributed to decreased osteoclast formation and bone resorption activity due to the increased serum levels of IFN-g. 13 Taken together, these data highlight an important role for DLX3 in bone; however, the studies above 12,13 provide contrasting results and emphasize that the function of DLX3 in the postnatal and adult skeleton has not yet been fully elucidated. To address the in vivo role of DLX3 in osteoblastogenesis, bone density, and remodeling in the appendicular skeleton, we generated conditional knockouts (cKOs) of Dlx3 in mesenchymal cells (Dlx3 Prx1-cKO ) and in osteogenic lineage cells (Dlx3 OCN-cKO ).…”