Figure legends
Figure 1. The histological sections of human deciduous molar germ from the cap-to-early bell stage. At embryonic stages of 12 weeks (A, late cap stage), 15 weeks (B, early bell stage), and 18 weeks (C, bell stage), the tooth germ gradually enlarges and differentiates. Scale bar, 100 μm. DP, dental papilla; IDE, inner dental epithelium; ODE, outer dental epithelium; DF, dental follicle; SR, stellate reticulum; SI, stellate intermedium; PEK, primary enamel knot; PA, preameloblast.
Figure 2. The overall identified proteins in different stages in human deciduous molar germ. (A) Venn diagram illustrating the overlapping proteins among the number of proteins identified in different stages of the human deciduous molar. (B) Inter-individual variation analysis of different stages. (C) The summary of high-coefficient of variation (CV) proteins and low-CV proteins in human tooth germ. (D) Results of PANTHER protein class illustrating overlapping proteins during the cap-to-bell transition in human embryonic tooth germ. (E) Venn diagram displaying the tissue-specific proteins of molar proteome compared with proteins in Human Protein Atlas (HPA) and Genotype-Tissue Expression project (GTEx) databases. Functional enrichment results and the protein are listed. (F) The temporal expression of proteins involved in the important developmental pathways retrieved from the common knowledge databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, and GeneCards).
Figure 3. The expression analyses of all identified proteins. (A) Clustering dendrogram of all proteins based on dissimilarity algorithm and assignment modules. (B) Module-trait correlation heatmap between clinical traits and modules. (C) Correlation scatter plot between protein significance and module membership in MEpaleturquoise and MEgreenyellow modules. (D) Functional enrichment analysis of proteins in MEpaleturquoise and MEgreenyellow modules. (E) Hub proteins expression heatmap. (F) UMAP plots showed distributions of dental cell subpopulations of public database (top); The bar charts (below) representing the cell ratios of different cell populations. (G) Average gene expression heatmap plot of different dental cell populations (top); Temporal gene expression in four types of dental cells (below).
Figure 4. The protein expression analyses of differentially expressed proteins (DEPs). (A) Fuzzy c-means clustering identifying five temporal expression patterns and the corresponding biological processes. TFs, transcription factors. (B) Top 20 proteins with the highest degree scores by Cytohubba algorithm. (C) Average expression heatmap plot of the top 20 proteins with five temporal expression patterns (top); Temporal gene expression of the top 20 proteins in different cell populations (below).
Figure 5. The validation of four DEPs (AFM, GJA1, PAX9, and KRT15) by IHC analysis. (A) The normalized mass-spectrometry intensity of DEPs from cap to early bell stage. (B) The representative images and relative quantitative value of IHC staining. Arrow indicated dental epithelial cells. Magnification, 200×. DEPs, differentially expressed proteins; AFM, Afamin; GJA1, Gap junction alpha-1; PAX9, Paired box 9; KRT15, keratin 15.