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.