International Journal of Oral Science (2012) 4, 7–13 2012 WCSS. All rights reserved 1674-2818/12 www.nature.com/ijos ORIGINAL ARTICLE Laser capture microdissection enables cellular and molecular studies of tooth root development Jian-Xun Sun1,2,*, Orapin V Horst3,*, Roger Bumgarner4, Bryce Lakely5, Martha J Somerman6 and Hai Zhang7 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. International Journal of Oral Science (2012) 4, 7–13; doi:10.1038/ijos.2012.15; published online 16 March 2012 Keywords: gene; laser capture microdissection; microarray; PCR; root INTRODUCTION Tooth formation proceeds through a series of highly orchestrated reciprocal signaling interactions between dental epithelial and mesen- chymal cells, termed epithelial–mesenchymal interactions (EMIs).1–5 Many genes/proteins involved in this process were identified and mapped temporally and spatially.6–7 In contrast to the crown formation, very few regulators of the root formation were identified.8–11 Work by our group and others suggests a role of epithelial signals in inducing the root development. The root forming cells such as cementoblasts (CB), dental follicle (DF) cells and other dental mesenchymal cells respond to enamel matrix proteins both in vitro and in vivo.11–18 Further support for a role of epithelial products in root development comes from tooth organ culture data and from investigation of genes expressed by Hertwig’s epithelial root sheath.17,19–20 The data from these studies suggest that genes expressed by epithelial cells during root development are significantly different from those expressed during early stages of crown development; how- ever, in depth studies to confirm these proposals, as well as knowledge as to specific similarities and/or differences in genes/proteins for crown vs. root development are minimal. To determine the role of specific genes during tooth development, gene knockout animal models are often used. Many of these animal models are lethal and thus are only suitable for examining early stages of tooth development, i.e., crown development, since in rodents, first molar crown development is almost complete prior to birth. By limiting gene ablation to specific tissues and thus allowing for longer lifespans, conditional gene knockout models have served some purposes; however, without having specific markers for targeting tooth root development, such models have limited value.21–22 Laser capture microdissection (LCM) has been used extensively to analyze gene expression profiles in various non-mineralized tis- sues23–24 and in the developing teeth at early stages of crown formation when the tooth is not highly mineralized.25–29 In mineralized tissue research, the difficulty in preparing intact unfixed, undecalcified and highly mineralized tissue sections (such as dentin and enamel) with well- preserved cellular structures and high-quality RNA for LCM is a com- mon obstacle. Over the past several years, we have optimized a technique for LCM of cells associated with the dento-alveolar bone complex. In this study, we aim to describe a new approach for tissue preparation and cell collection using LCM of postnatal day 7 (P7) and P14 mouse mandibles. This technique facilitates the use of LCM and microarrays to delineate regulatory mechanisms of the root development. Mouse incisors are continuously erupting and have all stages of differentiating cells, while their molars develop in a similar fashion 1State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China; 2Department of Periodontics, University of Washington, Seattle, WA, USA; 3Division of Endodontics, Department of Preventive and Restorative Dental Sciences, University of California at San Francisco, San Francisco, CA, USA; 4Department of Microbiology, University of Washington, Seattle, WA, USA; 5Department of Urology, University of Washington, Seattle, WA, USA; 6National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA and 7Department of Restorative Dentistry, University of Washington, Seattle, WA, USA * These authors contributed equally to this work. Correspondence: Dr H Zhang, 1959 NE Pacific St, Box 357456,Seattle, WA 98195, USA E-mail: haizhang@u.washington.edu Received 12 October 2011; Accepted 1 February 2012