3.4.Immunohistochemical staining of the selected proteins during embryonic tooth development
Due to the insufficient sequencing depth of single-cell data, the expression of AFM, one of the 24 screened proteins from WGCNA analysis, cannot be confirmed in scRNA-seq data. To validate the reliability of the high-throughput proteomic approach, we performed IHC staining on AFM, as well as on three representative markers of dental cells (Table S7 and Figure 5A). The expression of AFM, GJA1, PAX9 and KRT15 was validated via IHC analysis (Figure 5B).
The AFM immunostaining was widely and continuously observed in dental epithelial cells but could not be observed in dental mesenchymal cells after PCW15. Therefore, AFM may play a continuous role in the development of epithelial cells. GJA1 was observed in all tooth germ tissues, especially in IDE and SI/SR. During the cap-to-early bell stage, GJA1 localization in dental papilla gradually increased, thus increasing the IHC intensity of GJA1. The expression of PAX9 was detected in both dental epithelial and mesenchymal cells; its expression intensity was stronger in epithelium than in mesenchyme. In our study, KRT15-expressing cells were localized in the outer enamel epithelium and residual oral epithelium.
A comparison of the high-throughput proteomic and IHC analyses results revealed a similar expression pattern of AFM and GJA1. The intra-group CV values of these two proteins were relatively lower, and the repeatability were good. In contrast, the expression patterns of PAX9 and KRT15 were less consistent between the two methods. This result also suggests that both P -value and CV value should be considered for DEP screening.
Discussion
Given the significance of the cap-to-early bell stage during tooth morphogenesis, we analyzed the temporal patterns and identified the characteristic proteins during the transition from PCW12 to PCW18. Although the characteristic proteins obtained from different studies differed, their enrichment pathways were similar. Consistent with the previous proteomics research of miniature pig deciduous molar germ during the same phase, the biological processes include stem cell differentiation and classical developmental pathways such as Wnt and BMP signaling pathways were identified in our study[19]. Next, we performed a comparative proteomics analysis between the deciduous molar germ and postnatal human-derived dental stem cells. The proteins involving positive regulation of the locomotion pathway were up-regulated during the transformation stage from cap-to-early bell in our study. Earlier studies have reported that the characterized proteins in stem cells of exofoliated deciduous tooth are related to high cytoskeletal plasticity with cell adhesion[20]. The up-regulated pathways after dental papilla stem cell differentiation are involved in protein transport, cytoskeleton organization, extracellular matrix organization, intracellular protein transport, cell-cell adhesion, regulation of focal adhesion assembly, and vesicle docking[21]. These exocytosis-related pathways were also observed in our study.
Through WGCNA analysis, 24 hub proteins were screened for a high correlation with tooth development. Previous studies have reported that SPON1, AGO1, U2AF2, HMGB1, HMGB2, and PPP1R12A are closely related to stem cell proliferation and differentiation[22-24]. HMGB1 and HAPLN3 are involved in biomineralization. The expression of other hub proteins, such as RBP1 and CADM1 are restricted in the epithelium or the enamel knot[25-27]. To verify the origins of these proteins, transcriptomic analyses of dental cell populations are additionally adopted. The combined results provide the potential cell localization of hub proteins. The functions of these proteins are worthy for further investigation. Although scRNA-seq provides massive information from single cell population, due to the limitations of sequencing depth and sparse matrix, many valuable information may be missed. But this technology is a suitable supplement to proteomic research.
As a key component of cellular microenvironment, ECM provides supports for the development of tooth. Herein, ECM molecules including ECM glycoproteins, collagens, proteoglycans, ECM-affiliated proteins, regulators, and secreted factors, were explored[28]. Among the 320 identified ECM-associated proteins in human deciduous molar germ, 30 proteins were significantly up-regulated. These proteins included eight ECM glycoproteins (COMP, FBLN1, SLIT2, SMOC2, SPON1, TNFAIP6, VIT, and VWA2), two proteoglycans (HAPLN1 and HAPLN3), four ECM-affiliated proteins (FREM1, FREM2, MUC16, and SEMA4D), eight ECM regulators (CD109, F10, ITIH1, ITIH2, ITIH3, ITIH4, SERPINA3, and SERPINC1), and eight secreted factors (BMP7, INHBA, MDK, MST1, PTN, SCUBE1, SCUBE3, and SHH). The expression patterns of ECM-associated proteins will provide new insight into natural embryonic tooth development and new means to regenerate teeth using bioengineering methods.
In conclusion, we explored the biological process of human tooth morphogenesis from the cap-to-early bell stage using the healthy human deciduous molar proteomics.
Although our present study assessed the temporal protein patterns in a relative short period, the identified important biological processes and molecules will help researchers to mine the novel hypothesis of mineralized tissues development.