It is the long-term objective of this study to understand the process of root development at the morphological, cellular and molecular levels including the mechanisms underlying root dentin, cementum and periodontal ligament formation which leads to root attachment in the healthy periodontium. To accomplish this goal, we have focused our efforts in understanding the role of Hertwig?s epithelial root sheath (HERS) in this process. Towards this end, throughout the life of this grant, we have established several different immortal cell lines of the tissues associated with crown and root formation and using these cells lines we have provided evidence that HERS participate directly in the formation of acellular cementum. We have also shown that molecules secreted by HERS have a regulatory effect on the phenotype expressed by periodontal ligament cells. In these last years of support we continued our efforts to understand the process of root formation using a ?rootless? mice model resulting from the loss of function of the transcription factor Nuclear Factor I-C (NFI-C). The unique phenotype of these animals is the presence of overgrown maxillary incisors, lack of visible mandibular incisors and very short or absent molar roots. This phenotype indicates that the Nfi-c gene product is essential for the process of root formation. We also determined that there is formation of an abnormal shaped short root presenting with dentin dysplasia, which gets resorbed at later stages of development and there is marked delay in tooth eruption. The significance of Nfi-c in root development is validated by the recent report of a mutation in the NFI-C gene in a family affected by autosomal recessive radicular dentin dysplasia. In this competing renewal application we propose that Nfi-c regulates the initial stages of root development by regulating genes associated with HERS formation, proliferation and migration which in turn control root morphogenesis, odontoblast differentiation, dentin production and cementum formation. Furthermore, failure of HERS function will result in a cascade of root defects leading to root resorption. The following Specific Aims will test this hypothesis: 1) determine the mechanisms by which Nfi-c regulates HERS formation, proliferation and apical migration during root development;2) determine the mechanism by which HERS control odontoblast differentiation;3) determine the mechanism by which HERS controls cementum formation and 4) determine Nfi-c interactions and down-stream genes in root development. The data generated in this study will provide much needed information related to the process of root formation and the regulatory factors involved in this process. This data is of great health relevance to understand genetic defects resulting in root agenesis which result in premature tooth loss. Additionally, understanding root and periodontium development is critical to establish genetical and tissue engineering therapies for periodontium regeneration.