Daughters of the Enamel Organ: Development, Fate, and Function of the Stratum Intermedium, Stellate Reticulum, and Outer Enamel Epithelium

Abstract

The tooth enamel organ (EO) is a complex epithelial cell assembly involved in multiple aspects of tooth development, including amelogenesis. The present study focuses on the role of the nonameloblast layers of the EO, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium (OEE). The secretory stage stratum intermedium was distinguished by p63-positive epithelial stem cell marks, highly specific alkaline phosphatase labeling, as well as multiple desmosomes and gap junctions. At the location of the presecretory stage stellate reticulum, the pre-eruption EO prominently featured the papillary layer (PL) as a keratin immunopositive network of epithelial strands between tooth crowns and oral epithelium. PL cell strands contained numerous p63-positive epithelial stem cells, while BrdU proliferative cells were detected at the outer boundaries of the PL, suggesting that the stellate reticulum/PL epithelial cell sheath proliferated to facilitate an epithelial seal during tooth eruption. Comparative histology studies demonstrated continuity between the OEE and the general lamina of continuous tooth replacement in reptiles, and the outer layer of Hertwig's epithelial root sheath in humans, implicating the OEE as the formative layer for continuous tooth replacement and tooth root extension. Cell fate studies in organ culture verified that the cervical portion of the mouse molar EO gave rise to Malassez rest-like cell islands. Together, these studies indicate that the nonameloblast layers of the EO play multiple roles during odontogenesis, including the maintenance of several p63-positive stem cell reservoirs, a role during tooth root morphogenesis and tooth succession, a stabilizing function for the ameloblast layer, the facilitation of ion transport from the EO capillaries to the enamel layer, as well as safe and seamless tooth eruption.

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

“…Our previous studies had demonstrated the role of SI in enamel mineralization by the regulation of alkaline phosphatase activity in a mouse model ( Yoshizaki et al, 2014 , 2017 ). Some reports had earlier indicated that SI cells regulate ion transporters associated with ameloblast ( Liu H. et al, 2016 ). Solute carrier family member Slc4a4 (NBCe1) is complementarily expressed in ameloblasts and SI cells and has implicated functions in pH regulation during enamel mineralization ( Jalali et al, 2014 ), which is in agreement with our results ( Figure 2F ).…”

“…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 ). GO terms of the IEE/OEE cluster were enriched in cell mitosis ( Figure 2E ).…”

“…Interestingly, Krt15-positive cells have resistant cell property for oncogenesis ( Giroux et al, 2018 ; Moon et al, 2019 ). Given that OEE cells could provide progenitor cells ( Liu H. et al, 2016 ), Krt15-positive cells may play a role in the maintenance of stemness, preventing oncogenesis in OEE.…”

“…They may be classified into the following five types: ameloblast and its progenitor inner enamel epithelium (IEE), outer enamel epithelium (OEE), cuboidal cells surrounding the outer layer of the enamel organ, stratum intermedium (SI), squamous cells that cover ameloblast, and stellate reticulum (SR), having a spindle shape ( Krivanek et al, 2017 ). All these cell types are essential for proper enamel formation ( Liu H. et al, 2016 ). This classification is based on cell shape, and each cell type seems to play a distinct role; however, the transcriptomic characteristics and roles of OEE and SR are not yet fully understood.…”

Expression of new genes in vertebrate tooth development and p63 signaling

“…Keratins function in epithelial cell shape and cell-cell adhesion 41,42 as cited by Pan, et al 43 KRT15 may help maintain the shape of dental epithelium progenitor cells and facilitate their migration within the developing tooth organ to contribute to the stellate intermedium and, eventually, their differentiation into ameloblasts and cells of the stellate reticulum. 44 Like KRT15 ( Figure 5E,M,U), Krt8 was expressed in the dental epithelium and tooth organ ( Figure 4U-X). Krt8 expression is repressed by the p63 transcription factor 45 and induced when ΔN-p63 is down-regulated.…”