Distinct Gene Expression Patterns in Bones from Different Skeletal Sites

Objectives: We have sought to determine genetic differences that underlie phenotypic variation in bones from the cranial and appendicular skeleton. Such bones have differing functions and sensitivities to mechanical loads. Methods: 10 skeletally mature male black C57 mice were killed. Pooled bone samples were harvested from the parietal bones, mandible and ulna. In each case, external soft tissues were removed prior to snap-freezing in liquid nitrogen. The samples were powdered and transferred to QIAzol, homogenised and prepared for RNA extraction (RNeasy Mini Kit, Qiagen Ltd.). Sufficient quantities of extracted RNA allowed for duplicate samples to be prepared and hybridized in an Affymetrix mouse genome 430 2.0 gene array cassette. These cassettes represent over 34,000 well substantiated mouse genes. Data was analysed using GeneSpring GX7.3 (Agilent Technologies) software. Results: Preliminary analysis shows that the majority of expressed transcripts (17,456) are common to all sample sites. Some transcripts are peculiar to particular sites (calvaria; 1,173, mandible; 1,218, ulna; 1,247), whilst others are common to two sites (calvaria-mandible; 1,387, calvaria-ulna; 1,260, mandible–ulna; 609). These findings will be confirmed with real time PCR analysis. In addition, our data demonstrates that constitutive gene expression levels for certain genes are greater in the parietals and mandible compared with ulnae. Interestingly, genes coding cell growth/differentiation and cell adhesion proteins, for which constitutive expression is high in the mandible and parietals, correspond very closely to genes which are upregulated in ulnae following mechanical loading (Xing, J. Cell. Bio. 2005). Differences in constitutive gene expression might reflect the origin of the bone tissue, the role in the skeleton and, thus the phenotypic presentation. Conclusions: These data support the notion that regulation of calvarial and mandibular bone growth is not reliant on the mechanical loading stimuli known to engender functional adaptation in the appendicular skeleton. Supported by DoH grant.