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.