3.3.The temporal expression pattern of differentially expressed proteins during embryonic tooth development
The characteristic proteins in different stages were explored using one-way ANOVA and a total of 713 DEPs are screened (Table S6). Then the 713 DEPs were grouped into five groups with highly similar temporal expression patterns by using the same algorithm describe above (Figure 4A, left). The down-regulated DEPs in C1 and C5 are mainly involved in intracellular protein transport, microtubule cytoskeleton organization, and mitochondria translation. The up-regulated DEPs in C2 and C3 perform functions such as signal transduction and cell morphogenesis. The bipolar DEPs in C4 perform functions of epidermis development, differentiation, and negative regulation of endopeptidase activity. The differentially expressed molar TFs in various clusters were listed (Figure 4A, right). Concurrent with previous findings, the expression of MSX2 and SOX9 that participate in mesenchymal cell development and their levels gradually increases during cap to bell transition. NFIA, NFIB, NFIX and EBF3 which might participate in vesicle transport and neurogenic development show down-regulated during cap to bell transition.
Next, the interactive relationships of the key proteins in each temporal expression pattern were extracted using Cytoscape plugin (CytoHubba) (Figure 4B). To determine the main cell populations of these proteins function, the gene expression patterns of each cell population were compared from C1 to C5. Similar temporal mRNA expression patterns of cell populations and core functions were revealed (Figure 4C). The biological function of C3 cluster was related with embryonic development and all the single cell populations displayed the same temporal expression pattern. Although the transcripts of C3 were significantly increased in PCW15 stage, the up-regulated of proteins were not observed until PCW18 stage, demonstrating the discrepancies between mRNA and proteins. Besides, the biological function of C2 cluster was essential for vessel development. Accordingly, the endothelial cells and epithelial cells exhibited identical expression in mRNA and protein. The expression patterns of C4 cluster between epithelial cells and mesenchymal cells were completely opposite, suggesting that the interaction between epithelial and mesenchymal cells may be achieved through this group of molecules. Indeed, the epithelial-mesenchyme transition markers was identified for C4 cluster.