Tooth development is a complex process orchestrated by intricate gene regulatory networks, involving both odontogenic epithelium and ectomesenchyme. Six1, a pivotal transcription factor (TF), is involved in the development of the lower incisor. However, its precise role during incisor development and the molecular mechanisms underpinning its regulatory functions remain poorly understood. This study employs Six1 deletion mouse models to elucidate the critical regulatory role of Six1 in governing dental mesenchyme development. By performing single-cell RNA sequencing, we constructed a comprehensive transcriptome atlas of tooth germ development from the bud to bell stage. Our analyses suggest that the dental follicle and the dental papilla (DP) are differentiated from dental ectomesenchyme (DEM) and identify the key TFs underlying these distinct states. Notably, we show that Dlx1, Dlx2, and Dlx5 (Dlx1/2/5) may function as the key TFs that promote the formation of DP. We further show that the deletion of Six1 perturbs dental mesenchyme development by impeding the transitions from DEM to DP states. Importantly, SIX1 directly binds to the promoters of Dlx1/2/5 to promote their co-expression, which subsequently leads to widespread epigenetic and transcriptional remodeling. In summary, our findings unveil Six1\'s indispensable role in incisor development, offering key insights into TF-driven regulatory networks that govern dental mesenchyme cell fate transitions during tooth development.

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

OBJECTIVE: Fibroblast growth factors (FGFs) play pivotal roles in mediating interactions between dental epithelium and mesenchyme throughout tooth initiation and morphogenesis. This study aimed to elucidate the roles of FGF4 and FGF10 in the regulation of tooth development.
METHODS: In this study, we investigated spatiotemporal expression patterns of FGF4 and FGF10 in the third deciduous molars (DM3) of miniature pigs at the cap, early bell, and late bell stages. Pregnant miniature pigs were obtained, and the samples were processed for histological staining. Non-radioactive in situ hybridization, immunohistochemistry, and real-time PCR were used to detect mRNA and protein expression levels of FGF4 and FGF10.
RESULTS: FGF4 was expressed in the dental epithelium and mesenchyme at the cap stage. At the early bell stage, epithelial expression of FGF4 was reduced while mesenchymal expression got stronger. At the late bell stage, the FGF4 expression was restricted to the inner enamel epithelium (IEE) and differentiating odontoblasts. FGF10 was expressed intensely in both epithelium and mesenchyme at the cap stage. The expression of FGF10 was concentrated in the secondary enamel knots and surrounding mesenchyme at the early bell stage. FGF10 was weakly detected in the IEE by the late bell stage.
CONCLUSIONS: Our results indicated that FGF4 and FGF10 might have partially redundant functions in regulating epithelium morphogenesis. FGF4 may be involved in regulatory signaling cascades mediating interactions between the epithelium and mesenchyme. In addition, the downregulation of FGF10 expression may be associated with the cessation of mesenchymal cell proliferation and initiation of preodontoblast polarization.

The fibroblast growth factor (FGF) pathway plays an important role in epithelial-mesenchymal interactions during tooth development. Nevertheless, how the ligands, receptors, and antagonists of the FGF pathway are involved in epithelial-mesenchymal interactions remains largely unknown. Miniature pigs exhibit tooth anatomy and replacement patterns like those in humans and hence can serve as large animal models. The present study investigated the spatiotemporal expression patterns of critical genes encoding FGF ligands (FGF3, FGF4, FGF7, and FGF9), antagonists (SPRY2 and SPRY4) and receptors (FGFR1, FGFR2, and FGFR3) in the third deciduous molars of miniature pigs at the cap (embryonic day 40, E40), early bell (E50), and late bell (E60) stages. The results of in situ hybridization (ISH) with tyramide signal amplification and of qRT-PCR analysis revealed increased expression of FGF7, FGFR1, FGFR2, and SPRY4 in dental epithelium and of FGF7 and FGFR1 in mesenchyme from E40 to E50. In contrast, the results revealed decreased expression of FGF3, FGF4, FGF9, and FGFR3 in dental epithelium and of FGF4, FGF9, FGFR2, and FGFR3 in the mesenchyme from E40 to E60. Mesenchyme signals of FGF3, FGF4, FGF7, SPRY2, FGFR2, and FGFR3 were concentrated in the odontoblast layer from E50 to E60. The distinct expression patterns of these molecules indicated elaborate regulation during dental morphogenesis. Our results provide a foundation for further investigation into fine-tuning dental morphogenesis and odontogenesis by controlling interactions between dental epithelium and mesenchyme, thus promoting tooth regeneration in large mammals.

The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown.
A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos.
Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme.

Dentinogenesis, a formation of dentin by odontoblasts, is an essential process during tooth development. Bone morphogenetic proteins (BMPs) are one of the most crucial growth factors that contribute to dentin formation. However, it is still unclear how BMP signaling pathways regulate postnatal crown and root dentinogenesis. BMPs transduce signals through canonical Smad and non-Smad signaling pathways including p38 and ERK signaling pathways. To investigate the roles of Smad and non-Smad signaling pathways in dentinogenesis, we conditionally deleted Bmpr1a, which encodes the type 1A receptor for BMPs, to remove both Smad and non-Smad pathways in Osterix-expressing cells. We also expressed a constitutively activated form of Bmpr1a (caBmpr1a) to increase Smad1/5/9 signaling activity without altered non-Smad activity in odontoblasts. To understand the function of BMP signaling during postnatal dentin formation, Cre activity was induced at the day of birth. Our results showed that loss of BmpR1A in odontoblasts resulted in impaired dentin formation and short molar roots at postnatal day 21. Bmpr1a cKO mice displayed a reduction of dentin matrix production compared to controls associated with increased cell proliferation and reduced Osx and Dspp expression. In contrast, caBmpr1a mutant mice that show increased Smad1/5/9 signaling activity resulted in no overt tooth phenotype. To further dissect the functions of each signaling activity, we generated Bmpr1a cKO mice also expressing caBmpr1a to restore only Smad1/5/9 signaling activity. Restoring Smad activity in the compound mutant mice rescued impaired crown dentin formation in the Bmpr1a cKO mice; however, impaired root dentin formation and short roots were not changed. These results suggest that BMP-Smad signaling in odontoblasts is responsible for crown dentin formation, while non-Smad signaling may play a major role in root dentin formation and elongation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.

Innervation plays a key role in the development and homeostasis of organs and tissues of the orofacial complex. Among these structures, teeth are peculiar organs as they are not innervated until later stages of development. Furthermore, the implication of neurons in tooth initiation, morphogenesis and differentiation is still controversial. Co-cultures constitute a valuable method to investigate and manipulate the interactions of nerve fibers with their target organs in a controlled and isolated environment. Conventional co-cultures between neurons and their target tissues have already been performed, but these cultures do not offer optimal conditions that are closely mimicking the in vivo situation. Indeed, specific cell populations require different culture media in order to preserve their physiological properties. In this study we evaluate the usefulness of a microfluidics system for co-culturing mouse trigeminal ganglia and developing teeth. This device allows the application of specific media for the appropriate development of both neuronal and dental tissues. The results show that mouse trigeminal ganglia and teeth survive for long culture periods in this microfluidics system, and that teeth maintain the attractive or repulsive effect on trigeminal neurites that has been observed in vivo. Neurites are repealed when co-cultured with embryonic tooth germs, while postnatal teeth exert an attractive effect to trigeminal ganglia-derived neurons. In conclusion, microfluidics system devices provide a valuable tool for studying the behavior of neurons during the development of orofacial tissues and organs, faithfully imitating the in vivo situation.