Laser capture microdissection enables cellular and molecular studies of tooth root development

Epithelial–mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development. Meticulous laser excision of oral tissues has enabled researchers from the US and China to identify genes involved in tooth-root development. Better understanding of the spatial and temporal regulation of these genes could open the way for gum and tooth regenerative therapies. Led by Hai Zhang from the University of Washington,Seattle,the researchers used two complementary gene profiling approaches to identify genes that were differentially expressed in pure samples of five cell- and tissue-types excised from one- and two-week-old mice using the technique known as laser capture microdissection. The findings will help scientists to elucidate the carefully orchestrated molecular interactions between the epidermal and mesenchymal cell layers that control root formation,including the dramatic differentiation of soft tissues into enamel—the body's hardest tissue.