The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial–mesenchymal interactions

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Abstract

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial–mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.

Research highlights

► Bioengineered teeth regulated the contact area of epithelium and mesenchyme. ► The crown width is regulated by the contact area of the epithelium and mesenchyme. ► This regulation is associated with cell proliferation and Sonic hedgehog expression. ► The cusp number is correlated with the crown width of the bioengineered tooth. ► Cell proliferation and Shh expression areas regulate the tooth morphogenesis.

Introduction

All organs arise from their respective germs through reciprocal interactions between the epithelium and mesenchyme during organogenesis in the developing embryo [1], [2], [3], [4]. Organs develop according to predetermined programs, which include the regulation of their location, cell number and morphology. The induction of organ development at the appropriate future location requires both regional and genetic specificity [5]. It is well known in this regard that many cytokines, such as the fibroblast growth factor (FGF), hedgehog, Wnt, and transforming growth factor (TGF)/bone morphogenetic protein (BMP) families, play essential roles in epithelial and mesenchymal interactions during organogenesis [1], [2].

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, also develop through reciprocal epithelial and mesenchymal interactions [1], [2]. The number and morphology of the teeth in the tooth forming field, which are specified by the expression of homeobox genes in the underlying neural crest-derived mesenchyme in the embryonic jaw, have previously been determined during developmental process [3]. Tooth development begins with epithelium thickening and innervation of the underling mesenchyme [1], [2]. At the dental placode stage, the dental epithelium induces the condensation of the surrounding mesenchymal cells through the expression of signaling molecule genes such as Shh, Fgf8, Bmp4 and Wnt10b, which can induce the expression of a large number of transcription factors such as Msx1, Pax9 and Gli in the mesenchyme [2]. These interactions between these signaling molecules and transcription factors induce the formation of an enamel knot, which acts as a signaling center to coordinate tooth germ development [2]. Shh plays a particularly important role in tooth germ induction and formation, including the primary enamel knot formation, and thereafter functions in the growth and differentiation of epithelial cells into the ameloblast [6].

Following tooth germ formation, the epithelial and mesenchymal cells in the tooth germ differentiate into tooth-tissue forming cells and secrete hard tissues such as enamel dentin, cementum, and alveolar bone [7]. The tooth types that result, such as incisors (monocuspid) and molars (multicuspid), are thought to be regulated by regional gene expression which controls the tooth-forming region at the mesenchyme during embryonic development [3]. It has also been reported that the tooth type and morphology is determined by the balance of endogenous inhibitors and mesenchymal activator [8] and by regulatory mechanisms that operate in the tooth forming field [9]. Tooth morphology is defined by both the crown size and tooth length at the macro-morphology, and by the number and position of the cusp and roots at the micro-morphology [10]. Although the crown size, as a determinant of macro-patterning during tooth morphogenesis, is based on the reaction-diffusion model [10], the underlying molecular and cellular mechanisms, such as cell growth and cell movement, have remained unexplored. The regulation of the cusp number and position, which underlies the micro-patterning of the tooth, is thought to be closely involved in the formation of the secondary enamel knot. This is regulated spatiotemporally by the reciprocal activation and inhibition of cell proliferation in the epithelium and mesenchyme via the reaction-diffusion mechanism, and determines the cusp pattern formation through cell growth and movement [11], [12]. However, it remains to be undetermined how the regulation of cell proliferation and the underlying molecular mechanisms are involved in crown size determination through epithelial–mesenchymal interactions.

In our current study, we analyzed the mechanisms that determine the crown width and cusp number of a bioengineered tooth via the regulation of the contact area between the epithelial and mesenchymal cell layers. We provide evidence to suggest that the spatiotemporal regulation of epithelial cell proliferation and Shh expression in the tooth germ-epithelium is involved in determining the crown and cusp morphologies during tooth development.

Section snippets

Animals

C57BL/6 mice were purchased from SLC Inc., (Shizuoka, Japan). B6.Cg-Shhtm1(EGFP/cre)Cjt/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mouse care and handling complied with the NIH guidelines for animal research and all experimental protocols involving animals were approved by the Tokyo University of Science Animal Care and Use Committee.

Reconstitution of a bioengineered tooth germ from single cells

Molar tooth germs were dissected from the mandibles of ED14.5 mice in order to reconstitute a bioengineered tooth germ by a

The crown width of a bioengineered tooth correlates with the length of the contact area between the epithelial and mesenchymal cell layers

We first investigated whether the contact area between the epithelial and mesenchymal cell layers affect the eventual morphology, such as the crown width and cusp number, of a bioengineered tooth germ reconstituted from ED14.5 molar tooth germ-derived single cells using the organ germ method [13]. The bioengineered tooth germs, which were prepared using various contact lengths between the epithelial and mesenchymal cell layers, were reconstructed with a micro-syringe of a 0.330 μm inner diameter

Discussion

We demonstrate herein that the crown width of a bioengineered tooth is regulated by the contact area between the epithelial and mesenchymal cell layers and associates with cell proliferation and Shh expression in the inner enamel epithelium. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings also suggest that the spatiotemporal patterning of the cell proliferation and Shh expression areas in epithelium regulates

Acknowledgments

This work was partially supported by Health and Labour Sciences Research Grants from the Ministry of Health, Labour, and Welfare (No. 21040101), a Grant-in-Aid for Scientific Research in Priority Areas (No. 50339131), a Grant-in-Aid for Scientific Research (A) from Ministry of Education, Culture, Sports and Technology, Japan (all to T.T.).

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