Despite substantial advances towards understanding enamel and dentine formation, remarkably little is known about Hertwig's epithelial root sheath (HERS) differentiation, the induction of cementogensis and root formation. The major objective of this research project is to determine the molecular phenotype of HERS during initial root formation in hamster mandibular first molar development. We propose to test the hypothesis that following crown formation of the tooth organ, HERS cells synthesize and secrete non-collagenous enamel-related proteins which form intermediate cementum (IC) upon the root dentine surface. The following specific aims are designed to test our hypothesis: (i) identify and characterize IC proteins which share epitopes with specific domains found in enamel proteins; (ii) determine time-and position-restricted immunolocalization of IC proteins during molar root formation in situ, (iii) to examine the putative inductive activity of IC proteins in long-term tooth organ culture in vitro using serumless, chemically-defined medium; (iv) identify and characterize ICmRNA(s); (v) produce a cDNA library from HERS-derived mRNAs, and identify, isolate and characterize an ICcDNA; (vi) produce cRNA probes for in situ hybridization to map time-and position-restricted localization of IC gene transcripts during root development; and (vii) sequence the major ICcDNA and predict the amino acid sequence and secondary structure of this gene product(s). Methods employed include microdissection, protein extracts, one-and two-dimensional gel electrophoresis, immunotransfers, fluorography, amino acid composition and primary sequence analysis, production of oligopeptides and antibodies, organ culture, light and electron microscopy, indirect immunofluorescent microscopy, recombinant DNA techniques, sense and antisense cRNA probe production, in situ hybridization and nucleic acid sequencing. These studies use fetal, and postnatal hamster tooth organs.
