The Role Of Ndrg2Gene In Mouse Vertebral Development

The vertebrae-related disorders, such as the vertebralhyperosteogeny, spondylocostal dysostosis, and the protrusion ofintervertebral disc, are prevalent in the elderly worldwide. The underlyingmechanism remains unclear until now. Therefore, the investigations intothe vertebral development, including patterning and morphogenesis,would lend support for the therapy of these disorders. The vertebralskeletons consist of cervical, thoracic, lumbar, sacral and caudal vertebrae,which exhibit regionalized morphological characteristics inanterior-posterior (AP) direction. The vertebrae are derived from thesclerotome of somite mesoderm, which is periodically produced from thethe presomitic mesoderm (PSM). It is generally considered that thevertebral patterning and identity are globally determined by molecularmachinery of oscillating signaling and Hox genes in the PSM. Relativelittle is known about the regulators of the vertebral specification after somite segmentation. Here we presented that Ndrg2, a tumor suppressorgene, was dynamically expressed in PSM and the mesenchyme ofdifferentiating somites. In addition, it was also expressed duringendochondral ossification. To understand the role of Ndrg2gene in thetwo developmental processes, we performed in vitro and in vivo studiesthrough genetic approaches of transgenic and knockout mice.Ndrg2was mainly expressed in condensed mesenchymal stem cells(MSCs) during mouse endochondral ossification. When the MSCsundergo chondrogenic or osteogenic differentiation, Ndrg2was expressedin perichondrium cells which were bone progenitor cells. Forced Ndrg2expression in ATDC5and MC3T3E1delayed their osteogenicdifferentiation. In addition, over express Ndrg2in the limb of developingchicken embryo also delayed chondrocyte hypertrophy and boneformation of the distal digit. During somitegenesis, Ndrg2was observedin the presomitic mesoderm (PSM) as well as most of mesenchymal cellsof differentiating somites.To investigate the function of Ndrg2during vertebral developmentand endochondral ossification, we generated Ndrg2conditional knockoutmice. Homozygous mutant mice carrying a targeted deletion of Ndrg2didnot show any defects during endochondral bone formation. However,they exhibited vertebral homeotic transformations in thoracic, lumbar,and sacral regions. Osteoblast-specific (Col1a1-Cre3.6-kb) and chondrocyte-specific (Col2a1-Cre) Ndrg2knockout mice recapitulatedthe phenotype observed in Ndrg2-/-mice, suggesting that Ndrg2cell-autonomously regulates the patterning of axial skeleton in segmentedsomites rather than in the PSM. The effect of Ndrg2was dose dependent,as Ndrg2+/-mice had milder phenotype and a lower penetrance thanNdrg2-/-mice. To validate this point of view, we generated Col1a1(3.6kb)-Ndrg2and Col2a1-Ndrg2transgenic mice. Both Col1a1-Ndrg2andCol2a1-Ndrg2mice exhibited vertebral homeotic transformations whichwere distinct from the phenotype observed in Ndrg2-/-mice. Ndrg2-/-embryos showed alterations in expression levels of Hox genes insegmented somites, indicating that Ndrg2acts upstream of the Hox genesand that the functional role of Hox gene in segmented somites isnecessary for finally commitment somitic identity. Finally, we found thatboth Bmp4expression level and Bmp/Smad signaling activity wereelevated in Ndrg2-/-embryos.Our findings reveal Ndrg2as a novel regulator of vertebralpatterning and morphogenesis in differentiating somites. It instructsvertebral specification via Hox genes and Bmp signaling. Moreimportantly, our study indicates that the somitic identity is still plastic insegmented somite, and provides novel cues for the potential role of Hoxgenes in vertebral specification in segmented somites. It is different fromthe general point of view that the somitic patterning and identity are determined prior somite segmentation. Therefore our results shed newlights into the plasticity of segmented somite identity and the postsomticframework of vertebral patterning.