Plasticity within the niche ensures the maintenance of a Sox2+ stem cell population in the mouse incisor.

In mice, the incisors grow throughout the animal's life, and this continuous renewal is driven by dental epithelial and mesenchymal stem cells. Sox2 is a principal marker of the epithelial stem cells that reside in the mouse incisor stem cell niche, called the labial cervical loop, but relatively little is known about the role of the Sox2+ stem cell population. In this study, we show that conditional deletion of Sox2 in the embryonic incisor epithelium leads to growth defects and impairment of ameloblast lineage commitment. Deletion of Sox2 specifically in Sox2+ cells during incisor renewal revealed cellular plasticity that leads to the relatively rapid restoration of a Sox2-expressing cell population. Furthermore, we show that Lgr5-expressing cells are a subpopulation of dental Sox2+ cells that also arise from Sox2+ cells during tooth formation. Finally, we show that the embryonic and adult Sox2+ populations are regulated by distinct signalling pathways, which is reflected in their distinct transcriptomic signatures. Together, our findings demonstrate that a Sox2+ stem cell population can be regenerated from Sox2- cells, reinforcing its importance for incisor homeostasis.

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development.

Bmi1+ Progenitor Cell Dynamics in Murine Cornea During Homeostasis and Wound Healing.

The outermost layer of the eye, the cornea, is renewed continuously throughout life. Stem cells of the corneal epithelium reside in the limbus at the corneal periphery and ensure homeostasis of the central epithelium. However, in young mice, homeostasis relies on cells located in the basal layer of the central corneal epithelium. Here, we first studied corneal growth during the transition from newborn to adult and assessed Keratin 19 (Krt19) expression as a hallmark of corneal maturation. Next, we set out to identify a novel marker of murine corneal epithelial progenitor cells before, during and after maturation, and we found that Bmi1 is expressed in the basal epithelium of the central cornea and limbus. Furthermore, we demonstrated that Bmi1+ cells participated in tissue replenishment in the central cornea. These Bmi1+ cells did not maintain homeostasis of the cornea for more than 3 months, reflecting their status as progenitor rather than stem cells. Finally, after injury, Bmi1+ cells fueled homeostatic maintenance, whereas wound closure occurred via epithelial reorganization. Stem Cells 2018;36:562-573.

Suppression of epithelial differentiation by Foxi3 is essential for molar crown patterning.

Epithelial morphogenesis generates the shape of the tooth crown. This is driven by patterned differentiation of cells into enamel knots, root-forming cervical loops and enamel-forming ameloblasts. Enamel knots are signaling centers that define the positions of cusp tips in a tooth by instructing the adjacent epithelium to fold and proliferate. Here, we show that the forkhead-box transcription factor Foxi3 inhibits formation of enamel knots and cervical loops and thus the differentiation of dental epithelium in mice. Conditional deletion of Foxi3 (Foxi3 cKO) led to fusion of molars with abnormally patterned shallow cusps. Foxi3 was expressed in the epithelium, and its expression was reduced in the enamel knots and cervical loops and in ameloblasts. Bmp4, a known inducer of enamel knots and dental epithelial differentiation, downregulated Foxi3 in wild-type teeth. Using genome-wide gene expression profiling, we showed that in Foxi3 cKO there was an early upregulation of differentiation markers, such as p21, Fgf15 and Sfrp5. Different signaling pathway components that are normally restricted to the enamel knots were expanded in the epithelium, and Sostdc1, a marker of the intercuspal epithelium, was missing. These findings indicated that the activator-inhibitor balance regulating cusp patterning was disrupted in Foxi3 cKO. In addition, early molar bud morphogenesis and, in particular, formation of the suprabasal epithelial cell layer were impaired. We identified keratin 10 as a marker of suprabasal epithelial cells in teeth. Our results suggest that Foxi3 maintains dental epithelial cells in an undifferentiated state and thereby regulates multiple stages of tooth morphogenesis.

Dental Epithelial Stem Cells Express the Developmental Regulator Meis1.

MEIS1 is a key developmental regulator of several organs and participates in stem cell maintenance in different niches. However, despite the murine continuously growing incisor being a well described model for the study of adult stem cells, Meis1 has not been investigated in a dental context. Here, we uncover that Meis1 expression in the tooth is confined to the epithelial compartment. Its expression arises during morphogenesis and becomes restricted to the mouse incisor epithelial stem cell niche, the labial cervical loop. Meis1 is specifically expressed by Sox2+ stem cells, which give rise to all dental epithelial cell lineages. Also, we have found that Meis1 in the incisor is coexpressed with potential binding partner Pbx1 during both embryonic and adult stages. Interestingly, Meis2 is present in different areas of the forming tooth and it is not expressed by dental epithelial stem cells, suggesting different roles for these two largely homologous genes. Additionally, we have established the expression patterns of Meis1 and Meis2 during tongue, hair, salivary gland and palate formation. Finally, analysis of Meis1-null allele mice indicated that, similarly, to SOX2, MEIS1 is not essential for tooth initiation, but might have a role during adult incisor renewal.