Itm2a is a type II transmembrane protein with a BRICHOS domain. cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway. Introduction The development of mammalian organs is mediated through sequential and reciprocal epithelial-mesenchymal interactions. The development of mouse tooth germ, like many other organs, occurs by coordinated multi-step molecular interactions . These molecular interactions are associated with more than 300 genes, and were related to the initiation and morphogenesis of the tooth germ C. However, the precise mechanisms underlying the molecular interactions related to tooth germ development are still unclear. Therefore, it is important to identify the regulatory genes that orchestrate these molecular interactions between dental epithelial and mesenchymal cells. We previously performed buy AZD5423 cDNA subtraction between the mandibles on embryonic day 10.5 (E10.5) and E12.0 to investigate the regulatory genes associated with odontogenesis. Nine highly expressed positive clones from the E10. 5 mandible and five highly expressed positive clones from the E12.0 mandible were obtained by colony array screening . The expression of several of these genes is closely associated with the developing tooth germ C. Integral membrane protein 2a (Itm2a) was one of the highly expressed genes in the E12.0 mandible . The Itm2a gene was originally isolated by a cDNA library subtraction of cultivated murine mandibular condyles . The Itm2a protein and its related proteins, Itm2b and Itm2c, belong to the BRICHOS superfamily. These Itm2 proteins consist of four regions with distinct properties; the hydrophobic, linker, BRICHOS and C-terminal regions, as do other proteins in the BRICHOS superfamily , and are type II transmembrane proteins. Itm2a bears one buy AZD5423 BRICHOS domain at the C-terminal extracellular side , . The expression of Itm2c is restricted to the brain , whereas Itm2b is ubiquitously expressed . Itm2a is expressed in the developing muscle  and in chondrocytes of the resting zone of the murine growth plate . Itm2a is also expressed in mature odontoblasts as well as T-cells and hair follicles , . However, the function of Itm2a is so far unknown, except for its potential to regulate chondrogenic and myogenic differentiation C. In addition, only a few studies have so far addressed the intracellular localization of Itm2a protein. In the murine thymoma cell line, EL4, the Itm2a protein was observed in Rabbit polyclonal to Coilin large cytoplasmic vesicles, and then translocated to the plasma membrane in the activated cells , whereas Itm2a protein was detected in the nuclei of human buy AZD5423 uterine endometrial stromal sarcoma cells . Thus far, the function, expression pattern and intracellular localization of Itm2a mRNA and protein have not yet been fully elucidated. In this study, the expression patterns of Itm2a mRNA and protein were examined in the course of tooth germ development from initiation to root formation. Furthermore, an buy AZD5423 immunocytochemistry study was performed using cultured dental epithelial mDE6 cells to confirm the intracellular localization of Itm2a protein in the dental epithelial cells. We herein address the possible functional roles of Itm2a during tooth development based on these results. Materials and Methods Animals BALB/c embryos and postnatal mice were used in the study. The examined embryonic and postnatal stages were E10.5, E12, E13, E14, E15, E16, E17 and E18 after gestation and postnatal days 0 (PN0), PN1, PN2, PN3, PN4, PN5, PN10 and PN20. Adult BALB/c mice were obtained from Charles River Japan Inc. (Yokohama, Japan). All experimental procedures using mice were approved by the Animal Care.