NFI-C Protein Expression Associated with Tooth Root Formation

Nuclear factor I-C (NFI-C) is a critical transcription factor for root formation. Previous studies in our laboratory have shown three NFI-C isoforms expressed in dental tissues (Lamani et al., 2008). Objectives: Generate/characterize a polyclonal anti-peptide human/mouse NFI-C isoform specific antibody; determine the temporal-spatial expression pattern of NFI-C during odontogenesis; and characterize isoform expression profiles. Methods: Mouse tissues at key stages of crown/root development were collected, fixed with demineralization, paraffin embedded and sectioned for immunohistochemistry. Human dental cells derived from enamel organ epithelium (EOE), periodontal ligament (PDL), and dental pulp apex/follicle (DF) were grown in supplemented medium under established conditions. Cells were fixed for immunohistochemistry or lysed for total protein. A rabbit polyclonal anti-peptide antibody raised against a conserved mouse/human C-terminus sequence unique to the NFI-C2 isoform was produced, titered and analyzed for specificity by ELISA and Western blotting. Lastly, the NFI-C2 antibody was compared to commercially available NFI-C antibodies (Santa Cruz, Abcam, and Abnova) by Western blot and immunoprecipitation. Results: The polyclonal anti-NFI-C2 antibody specificity was confirmed by ELISA with a titer of 1:10K. The recognition patterns of the NFI-C antibodies were found to differ. Immunostaining showed differential nuclear and cytosolic localization: one antibody preferentially recognized NFI-C in the ER/Golgi. The expression pattern of NFI-C in mouse developing teeth showed staining within both epithelial and mesenchymal tissues with high levels in the odontoblasts and ameloblasts. Conclusions: This study shows production of specific NFI-C2 peptide antibody; the temporal/spatial expression pattern of NFI-C protein during tooth formation for the first time; and confirmation of various isoforms in dental tissues. This data suggests a broader role for NFI-C during tooth formation than limited to root and periodontal ligament development. This data provides a foundation for understanding the molecular mechanisms involved in normal tooth and root development. Support: UAB-IOHR/NIDCR-F30DE018080/ProDoc-CAPES (ACA; Brazil).