The transcription factor AmeloD stimulates epithelial cell motility essential for tooth morphology

Abstract

The development of ectodermal organs, such as teeth, requires epithelial-mesenchymal interactions. Basic helixloop-helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD-knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD-KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells, and it may cause inhibition of the cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn-KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial-mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development. Ectodermal organs, such as teeth, all display a need for epithelial-mesenchymal interactions for their development. Tooth development is, in fact, a good model for understanding the mechanism of ectodermal development because it has welldefined stages and distinctive differentiated cell types (1). In the mouse, the morphogenesis of the molars is divided into four stages: the initiation stage (embryonic day 11.5 (E11.5)), 2 bud stage (E13.5), cap stage (E15.5), and bell stage (E17.5). At the initiation stage, the dental epithelium starts to thicken and invades into the mesenchymal region. This invagination process forms the tooth bud, and the dental epithelium condenses at the bud stage. After the cap stage, the dental epithelial stem cells differentiate into various cell types to form the enamel organ: the inner enamel epithelium (IEE), outer enamel epithelium, stratum intermedium (SI), and stellate reticulum. The IEE cells are ameloblast progenitor cells, a unique cell population in the proliferation stage as they express proliferation markers but do not express E-cadherin, a negative regulator of cell division and migration (2, 3). The IEE cells actively proliferate and migrate to form a correctly sized enamel organ. After proliferation, the IEE cells differentiate into enamel-secreting ameloblasts. Proliferative IEE cells persist near the apical tip of the

Single-Cell RNA-Sequencing From Mouse Incisor Reveals Dental Epithelial Cell-Type Specific Genes

“…OEE cells are known to contribute to root formation by constituting Hertwig’s epithelial root sheath. During tooth development, cell proliferation markers such as Ki67 are strongly positive in IEE ( Chiba et al, 2019 ); however, OEE proliferates more actively than IEE during the formation of Hertwig’s epithelial root sheath ( Yokohama-Tamaki et al, 2006 ). Also, in pre-eruption molars, OEE seems to be potent for proliferation ( Liu H. et al, 2016 ).…”

“…P1 and P3 mouse heads were embedded in paraffin, sectioned, and subjected to immunofluorescence, as described previously ( Chiba et al, 2019 ). The primary antibodies of AMEL (Abcam, 1:200), NOTCH2 (Cell Signaling Technology, 1:200), PTTG1 (Abcam, 1:100), CLDN10 (Thermo Fisher Scientific, 1:200), ATF3 (Abcam, 1:100), and KRT15 (Abcam, 1:200) were used for immunostaining.…”

“…For a better understanding of the molecular mechanism during tooth development, we had previously identified several genes preferentially expressed in ameloblast lineage and had analyzed their roles in tooth development ( Saito et al, 2015 ; Liu J. et al, 2016 ; Miyazaki et al, 2016 ; Nakamura et al, 2016 ; Arai et al, 2017 ; Han et al, 2018 ; Nakamura et al, 2020 ). We had also identified novel genes from tooth germ complementary DNA library, namely Ameloblastin ( Ambn ), Epiprofin ( Epfn ), and AmeloD ( Dhamija et al, 1999 ; Nakamura et al, 2004 ; He et al, 2019 ), and each of these knockout mice models showed severe enamel hypoplasia ( Fukumoto et al, 2004 ; Nakamura et al, 2008 , 2017 ; Aurrekoetxea et al, 2016 ; Chiba et al, 2019 ). Currently, ameloblast is the best-characterized cell type of dental epithelium and plays the most important role in enamel formation.…”

“…DESCs commit cell fate into ameloblast lineages and differentiate into IEE cells, which proliferate and migrate to increase the size of tooth germ during the proliferation stage ( Chiba et al, 2019 ). Soon after, IEE cells exit the cell cycle and become polarized pre-ameloblasts.…”

Expression Patterns of Claudin Family Members During Tooth Development and the Role of Claudin-10 (Cldn10) in Cytodifferentiation of Stratum Intermedium

“…Cldn10b-overexpressing SF2 cells showed a significant upregulation of Alpl expression, whereas Notch1 expression was downregulated ( Figure 4G). Expression of the IEE marker gene, AmeloD (Chiba et al, 2019;He et al, 2019), and ameloblast marker gene, Ambn, did not exhibit any notable changes ( Figure 4G). These results suggest that Cldn10b promotes SI cell differentiation in dental epithelium.…”