Quantitative proteomic analysis of deciduous molars during cap to bell transition in miniature pig

Taking advantage of genetic manipulation tools and accessibility, almost all molecular knowledge on vertebrate tooth development was obtained from rodent models that only have one dentition in their entire lives. Whether the tooth development in other vertebrates such as swine or human follows the same rules remains elusive. Rodent dentitions differ considerably from human dentitions, therefore limiting the application of knowledge from rodent tooth to human tooth. Signal-mediated communication between cells and complex gene and protein regulatory networks are key components of tooth development. By combining isobaric tandem mass tag (TMT) labeling with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technology, we constructed the proteomic profile of deciduous molars at embryonic days 40 and 50 in miniature pig (Sus scrofa). During the ten days of prenatal development of the miniature pig, the morphology of the lower deciduous molar moves from the early cap to the bell stage. Thus, we identified proteins that are associated with these developing stages and identified differentially regulated proteins (DRPs) that are potential or novel drivers of tooth morphogenesis. Three candidate proteins were validated via qRT-PCR, western blotting analysis, and the location of those proteins in tooth germ were observed by immunohistochemical staining. Multiple signaling pathways and protein interaction network revealed potential mechanisms of early tooth programming in a large mammal. Bioinformatic analysis also showed that cross interaction of Wnt and Sonic hedgehog pathways may play a key role in deciduous development during cap to bell transition in miniature pig. Significance: We performed the most comprehensive study of the whole tooth germ proteome in mammals to date. The high-throughput proteomic analysis identifies differentially regulated proteins and pathways that will help elucidate the mechanisms of tooth development.