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The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation. [ABSTRACT FROM AUTHOR]

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Here we generate a differentiation protocol based on this sci-RNA-seq analysis to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.

Summary: TSPEAR variants cause autosomal recessive ectodermal dysplasia (ARED) 14. The function of TSPEAR is unknown. The clinical features, the mutation spectrum, and the underlying mechanisms of ARED14 are poorly understood. Combining data from new and previously published individuals established that ARED14 is primarily characterized by dental anomalies such as conical tooth cusps and hypodontia, like those seen in individuals with WNT10A-related odontoonychodermal dysplasia. AlphaFold-predicted structure-based analysis showed that most of the pathogenic TSPEAR missense variants likely destabilize the β-propeller of the protein. Analysis of 100000 Genomes Project (100KGP) data revealed multiple founder TSPEAR variants across different populations. Mutational and recombination clock analyses demonstrated that non-Finnish European founder variants likely originated around the end of the last ice age, a period of major climatic transition. Analysis of gnomAD data showed that the non-Finnish European population TSPEAR gene-carrier rate is ∼1/140, making it one of the commonest AREDs. Phylogenetic and AlphaFold structural analyses showed that TSPEAR is an ortholog of drosophila Closca, an extracellular matrix-dependent signaling regulator. We, therefore, hypothesized that TSPEAR could have a role in enamel knot, a structure that coordinates patterning of developing tooth cusps. Analysis of mouse single-cell RNA sequencing (scRNA-seq) data revealed highly restricted expression of Tspear in clusters representing enamel knots. A tspeara−/−;tspearb−/− double-knockout zebrafish model recapitulated the clinical features of ARED14 and fin regeneration abnormalities of wnt10a knockout fish, thus suggesting interaction between tspear and wnt10a. In summary, we provide insights into the role of TSPEAR in ectodermal development and the evolutionary history, epidemiology, mechanisms, and consequences of its loss of function variants.

Background: Enamel knots and Hertwig epithelial root sheath (HERS) regulate the growth and folding of the dental epithelium, which subsequently determines the final form of tooth crown and roots. We would like to investigate the genetic etiology of seven patients affected with unique clinical manifestations, including multiple supernumerary cusps, single prominent premolars, and single-rooted molars. Methods: Oral and radiographic examination and whole-exome or Sanger sequencing were performed in seven patients. Immunohistochemical study during early tooth development in mice was performed. Results: A heterozygous variant (c. 865A>G; p.Ile289Val) in CACNA1S was identified in all the patients, but not in an unaffected family member and control. Immunohistochemical study showed high expression of Cacna1s in the secondary enamel knot. Conclusions: This CACNA1S variant seemed to cause impaired dental epithelial folding; too much folding in the molars and less folding in the premolars; and delayed folding (invagination) of HERS, which resulted in single-rooted molars or taurodontism. Our observation suggests that the mutation in CACNA1S might disrupt calcium influx, resulting in impaired dental epithelium folding, and subsequent abnormal crown and root morphology. [ABSTRACT FROM AUTHOR]

Background: Enamel knots and Hertwig epithelial root sheath (HERS) regulate the growth and folding of the dental epithelium, which subsequently determines the final form of tooth crown and roots. We would like to investigate the genetic etiology of seven patients affected with unique clinical manifestations, including multiple supernumerary cusps, single prominent premolars, and single-rooted molars. Methods: Oral and radiographic examination and whole-exome or Sanger sequencing were performed in seven patients. Immunohistochemical study during early tooth development in mice was performed. Results: A heterozygous variant (c. 865A>G; p.Ile289Val) in CACNA1S was identified in all the patients, but not in an unaffected family member and control. Immunohistochemical study showed high expression of Cacna1s in the secondary enamel knot. Conclusions: This CACNA1S variant seemed to cause impaired dental epithelial folding; too much folding in the molars and less folding in the premolars; and delayed folding (invagination) of HERS, which resulted in single-rooted molars or taurodontism. Our observation suggests that the mutation in CACNA1S might disrupt calcium influx, resulting in impaired dental epithelium folding, and subsequent abnormal crown and root morphology.

Enamel knot (EK) is known to be a central organ in tooth development, especially for cusp patterning. To trace the exact position and movement among the inner dental epithelium (IDE) and EK cells, and to monitor the relationship between the EK and cusp patterning, it is essential that we understand the cell cycle status of the EK in early stages of tooth development. In this study, thymidine analogous (IdU, BrdU) staining was used to evaluate the cell cycle phase of the primary EK at the early casp stage (E13.0) and the gerbil embryo (E19) in a developing mouse embryo. The centerpiece of this study was to describe the cell cycle phasing and sequencing during proliferation in the IDE according to the expression of IdU and BrdU following their injection at calculated time points. The interval time between IdU injection and BrdU injection was set at 4 h. As a result, the cell cycle in the IDE of the mouse and gerbil was found to be synchronous. Conversely, the cell cycle in primary EKs of mice was much longer than that of the IDE. Therefore, the difference of cell cycle of the IDE and the EK is related to the diversity of cusp patterning and would provide a new insight into tooth morphogenesis. [ABSTRACT FROM AUTHOR]

Vangl2, one of the core components of the planar cell polarity (PCP) pathway, has an important role in the regulation of morphogenesis in several tissues. Although the expression of Vangl2 has been detected in the developing tooth, its role in tooth morphogenesis is not known. In this study, we show that Vangl2 is expressed in the inner dental epithelium (IDE) and in the secondary enamel knots (SEKs) of bell stage tooth germs. Inhibition of Vangl2 expression by siRNA treatment in in vitro-cultured tooth germs resulted in retarded tooth germ growth with deregulated cell proliferation and apoptosis. After kidney transplantation of Vangl2 siRNA-treated tooth germs, teeth were observed to be small and malformed. We also show that Vangl2 is required to maintain the proper pattern of cell alignment in SEKs, which maybe important for the function of SEKs as signaling centers. These results suggest that Vangl2 plays an important role in the morphogenesis of teeth. [ABSTRACT FROM AUTHOR]

TSPEAR variants cause autosomal recessive ectodermal dysplasia (ARED) 14. The function of TSPEAR is unknown. The clinical features, the mutation spectrum, and the underlying mechanisms of ARED14 are poorly understood. Combining data from new and previously published individuals established that ARED14 is primarily characterized by dental anomalies such as conical tooth cusps and hypodontia, like those seen in individuals with WNT10A -related odontoonychodermal dysplasia. AlphaFold-predicted structure-based analysis showed that most of the pathogenic TSPEAR missense variants likely destabilize the β-propeller of the protein. Analysis of 100000 Genomes Project (100KGP) data revealed multiple founder TSPEAR variants across different populations. Mutational and recombination clock analyses demonstrated that non-Finnish European founder variants likely originated around the end of the last ice age, a period of major climatic transition. Analysis of gnomAD data showed that the non-Finnish European population TSPEAR gene-carrier rate is ∼1/140, making it one of the commonest AREDs. Phylogenetic and AlphaFold structural analyses showed that TSPEAR is an ortholog of drosophila Closca , an extracellular matrix-dependent signaling regulator. We, therefore, hypothesized that TSPEAR could have a role in enamel knot, a structure that coordinates patterning of developing tooth cusps. Analysis of mouse single-cell RNA sequencing (scRNA-seq) data revealed highly restricted expression of Tspear in clusters representing enamel knots. A tspeara -/- ; tspearb -/- double-knockout zebrafish model recapitulated the clinical features of ARED14 and fin regeneration abnormalities of wnt10a knockout fish, thus suggesting interaction between tspear and wnt10a. In summary, we provide insights into the role of TSPEAR in ectodermal development and the evolutionary history, epidemiology, mechanisms, and consequences of its loss of function variants., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: The gene regulatory network involved in tooth morphogenesis has been extremely well described in mammals and its modeling has allowed predictions of variations in regulatory pathway that may have led to evolution of tooth shapes. However, very little is known outside of mammals to understand how this regulatory framework may also account for tooth shape evolution at the level of gnathostomes. In this work, we describe expression patterns and proliferation/apoptosis assays to uncover homologous regulatory pathways in the catshark Scyliorhinus canicula. Results: Because of their similar structural and developmental features, gene expression patterns were described over the four developmental stages of both tooth and scale buds in the catshark. These gene expression patterns differ from mouse tooth development, and discrepancies are also observed between tooth and scale development within the catshark. However, a similar nested expression of Shh and Fgf suggests similar signaling involved in morphogenesis of all structures, although apoptosis assays do not support a strictly equivalent enamel knot system in sharks. Similarities in the topology of gene expression pattern, including Bmp signaling pathway, suggest that mouse molar development is more similar to scale bud development in the catshark. Conclusions: These results support the fact that no enamel knot, as described in mammalian teeth, can be described in the morphogenesis of shark teeth or scales. However, homologous signaling pathways are involved in growth and morphogenesis with variations in their respective expression patterns. We speculate that variations in this topology of expression are also a substrate for tooth shape evolution, notably in regulating the growth axis and symmetry of the developing structure. [ABSTRACT FROM AUTHOR]

The shape of an individual tooth crown is primarily determined by the number and arrangement of its cusps, i.e., cusp patterning. Enamel knots that appear in the enamel organ during tooth morphogenesis have been suggested to play important roles in cusp patterning. Animal model studies have shown that the Hippo pathway effector Yap has a critical function in tooth morphogenesis. However, the role of the Hippo pathway/Yap in cusp patterning has not been well documented and its specific roles in tooth morphogenesis remain unclear. Here, we provide evidence that Yap is a key mediator in tooth cusp patterning. We demonstrate a correlation between Yap localization and cell proliferation in developing tooth germs. We also show that, between the cap stage and bell stage, Yap is crucial for the suppression of the primary enamel knot and for the patterning of secondary enamel knots, which are the future cusp regions. When Yap expression is stage-specifically knocked down during the cap stage, the activity of the primary enamel knot persists into the bell-stage tooth germ, leading to ectopic cusp formation. Our data reveal the importance of the Hippo pathway/Yap in enamel knots and in the proper patterning of tooth cusps. [ABSTRACT FROM AUTHOR]

The activation of Wingless-type (Wnt)/β-catenin signaling is of fundamental importance in organogenesis. Wnt signaling is also known to regulate signaling crucial for tooth development. However, the underlying mechanism of Wnt activation or inhibition remains largely unknown. Here, we demonstrate that Wnt inhibitory factor 1 (Wif1), a secreted Wnt antagonist, localizes to the dental epithelium and mesenchyme during early tooth development. Specifically, Wif1 is strictly expressed in the enamel knot at embryonic day 14.5 (E14.5) and E16.5. The functional significance of Wif1 during tooth morphogenesis remains to be clarified. Our results reveal that the promotion of apoptosis by the knockdown of Wif1 leads to a delay in an early event during tooth development. This study therefore provides novel insights into the role of Wif1 and validates Wif1 as a potent target in WNT signaling during tooth development. We suggest that the enamel knots are central regulators of tooth development. Furthermore, Wif1 localizes to the enamel knot in which Wif1 regulates apoptosis by mediating and regulating Wnt-β-catenin signaling. Thus, Wif1 plays an essential role in tooth development. [ABSTRACT FROM AUTHOR]

Adenomatosis polyposis coli downregulated 1 (APCDD1), a negative regulator of Wnt signaling, was examined to understand detailed mechanisms underlying Wnt signaling tooth development. In situ hybridization showed that Apcdd1 was expressed in the condensed mesenchyme at the bud stage, and in the inner enamel epithelium (IEE), including enamel knot (EK) at the cap stage. In vitro organ cultivation by using Apcdd1 antisense oligodeoxynucleotides was performed at E13.5 for 2 days to define the developmental functions of APCDD1 during tooth development. Analysis of histogenesis and cellular events such as cell adhesion, proliferation, apoptosis and epithelial rearrangement after Apcdd1 knockdown showed altered morphogenesis of the tooth germ with decreased cell proliferation and altered localization of cell adhesion molecules. Actin filament staining and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) labeling of IEE cells showed that Apcdd1 knockdown enhanced epithelial rearrangement in the IEE and EK. To understand the precise signaling regulations of Apcdd1, we evaluated the altered expression patterns of signaling molecules, related with Wnt and enamel knot signalings using RT-qPCR. Tooth germs at cap stage were transplanted into the kidney capsules and were allowed to develop into calcified teeth for 3 weeks. Apcdd1 knockdown increased the number of ectopic cusps on the mesial side of the tooth. Our results suggested that APCDD1 modulates the gene expression of Wnt- and EK-related signaling molecules at the cap stage of tooth development, and is involved in tooth cusp patterning by modulating the epithelial rearrangement in the IEE. [ABSTRACT FROM AUTHOR]

Background: The mouse embryonic mandible comprises two types of tooth primordia in the cheek region: progressive tooth primordia of prospective functional teeth and rudimentary tooth primordia in premolar region - MS and R2. Mice lacking Sprouty genes develop supernumerary tooth in front of the lower M1 (first molar) primordium during embryogenesis. We focused on temporal-spatial dynamics of Sonic Hedgehog expression as a marker of early odontogenesis during supernumerary tooth development. Results: Using mouse embryos with different dosages of Spry2 and Spry4 genes, we showed that during the normal development of M1 in the mandible the sooner appearing Shh signaling domain of the R2 bud transiently coexisted with the later appearing Shh expression domain in the early M1 primordium. Both domains subsequently fused together to form the typical signaling center representing primary enamel knot (pEK) of M1 germ at embryonic day (E) 14.5. However, in embryos with lower Spry2;Spry4 gene dosages, we observed a non-fusion of original R2 and M1 Shh signaling domains with consequent formation of a supernumerary tooth primordium from the isolated R2 bud. Conclusions: Our results bring new insight to the development of the first lower molar of mouse embryos and define simple tooth unit capable of individual development, as well as determine its influence on normal and abnormal development of the tooth row which reflect evolutionarily conserved tooth pattern. Our findings contribute significantly to existing knowledge about supernumerary tooth formation. [ABSTRACT FROM AUTHOR]

The integument forms a number of different types of mineralized element, including dermal denticles, scutes, ganoid scales, elasmoid scales, fin rays and osteoderms found in certain fish, reptiles, amphibians and xenarthran mammals. To this list can be added teeth, which are far more widely represented and studied than any of the other mineralized elements mentioned above, and as such can be thought of as a model mineralized system. In recent years the focus for studies on tooth development has been the mouse, with a wealth of genetic information accrued and the availability of cutting edge techniques. It is the mouse dentition that this review will concentrate on. The development of the tooth will be followed, looking at what controls the shape of the tooth and how signals from the mesenchyme and epithelium interact to lead to formation of a molar or incisor. The number of teeth generated will then be investigated, looking at how tooth germ number can be reduced or increased by apoptosis, fusion of tooth germs, creation of new tooth germs, and the generation of additional teeth from existing tooth germs. The development of mineralized tissue will then be detailed, looking at how the asymmetrical deposition of enamel is controlled in the mouse incisor. The continued importance of epithelial–mesenchymal interactions at these later stages of tooth development will also be discussed. Tooth anomalies and human disorders have been well covered by recent reviews, therefore in this paper we wish to present a classical review of current knowledge of tooth development, fitting together data from a large number of recent research papers to draw general conclusions about tooth development. [ABSTRACT FROM AUTHOR]

Abstract: Tmem16a, Tmem16c, Tmem16f, Tmem16h and Tmem16k belong to the newly identified Tmem16 gene family encoding eight-pass transmembrane proteins. We have analyzed the expression patterns of these genes during mouse cephalic development. In the central nervous system, Tmem16a transcripts were abundant in the ventricular neuroepithelium, whereas the other Tmem16 family members were readily detectable in the subventricular zone and differentiating fields. In the rostral spinal cord, Tmem16f expression was highest in the motor neuron area. In the developing eye, the highest amounts of Tmem16a transcripts were detected in the lens epithelium, hyaloid plexus and outer layer of the retina, while the other family members were abundant in the retinal ganglionic cell layer. Interestingly, throughout development, Tmem16a expression in the inner ear was robust and restricted to a subset of cells within the epithelium, which at later stages formed the organ of Corti. The stria vascularis was particularly rich in Tmem16a and Tmem16f mRNA. Other sites of Tmem16 expression included cranial nerve and dorsal root ganglia, meningeal precursors and the pituitary. Tmem16c and Tmem16f transcripts were also patent in the submandibular autonomic ganglia. A conspicuous feature of Tmem16a was its expression along the walls of blood vessels as well as in cells surrounding the trigeminal and olfactory nerve axons. In organs developing through epithelial–mesenchymal interactions, such as the palate, tooth and tongue, the above five Tmem16 family members showed interesting dynamic expression patterns as development proceeded. Finally and remarkably, osteoblasts and chondrocytes were particularly loaded with Tmem16a, Tmem16c and Tmem16f transcripts. [Copyright &y& Elsevier]

Abstract: The heparin binding molecules MK and HB-GAM are involved in the regulation of growth and differentiation of many tissues and organs. Here we analyzed the expression of MK and HB-GAM in the developing mouse incisors, which are continuously growing organs with a stem cell compartment. Overlapping but distinct expression patterns for MK and HB-GAM were observed during all stages of incisor development (initiation, morphogenesis, cytodifferentiation). Both proteins were detected in the enamel knot, a transient epithelial signaling structure that is important for tooth morphogenesis, and the cervical loop where the stem cell niche is located. The functions of MK and HB-GAM were studied in dental explants and organotypic cultures in vitro. In mesenchymal explants, MK stimulated HB-GAM expression and, vice-versa, HB-GAM upregulated MK expression, thus indicating a regulatory loop between these proteins. BMP and FGF molecules also activated expression of both cytokines in mesenchyme. The proliferative effects of MK and HB-GAM varied according to the mesenchymal or epithelial origin of the tissue. Growth, cytodifferentiation and mineralization were inhibited in incisor germs cultured in the presence of MK neutralizing antibodies. These results demonstrate that MK and HB-GAM are involved in stem cells maintenance, cytodifferentiation and mineralization processes during mouse incisor development. [Copyright &y& Elsevier]

Abstract: Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation. [Copyright &y& Elsevier]

During molar development from the cap to bell stage, the morphology of the enamel knots, inner dental epithelium, and epithelial-mesenchymal junction dynamically changes, leading to the formation of multiple cusps. To study the basic histological features of this morphogenetic change, we have investigated the cell arrangement, mitosis, and apoptosis simultaneously in the developing first lower molar of the mouse by means of BrdU injection and immunostaining for P-cadherin, BrdU, and single-stranded DNA. At the typical cap stage, the primary enamel knot shows a characteristic cell arrangement, absence of mitosis, and abundant apoptosis, but also actively dividing cells at its lateral margins. Two secondary enamel knots then appear in the anterior part of the tooth germ. One is completely non-proliferating, whereas the other contains dividing cells, indicating asymmetrical growth of the inner dental epithelium. From this transitional stage to the early bell stage, additional minor BrdU-negative domains appear, and at the same time, the cell arrangement in the inner dental epithelium rapidly changes to show regional differences. Comparisons between the histology and the distribution of BrdU-positive cells have revealed that both the regionally different cell rearrangement and the differential cell proliferation in the enamel knots and inner dental epithelium probably play a significant role in multiple cusp formation. [ABSTRACT FROM AUTHOR]

The enamel knot (EK), which is located in the center of bud and cap stage tooth germs, is a transitory cluster of non-dividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps by inducing secondary EKs. The morphological, cellular, and molecular events leading to the relationship between the primary and secondary EKs have not been described clearly. This study investigated the relationship between the primary and secondary EKs in the maxillary and mandibular first molars of mice. The location of the primary EK and secondary EKs was investigated by chasing Fgf4 expression patterns in tooth germ at some intervals of in vitro culture, and the relationship between the primary EK and secondary EK was examined by tracing the primary EK cells in the E13.5 tooth germs which were frontally half sliced to expose the primary EK. After 48 hr, the primary EK cells in the sliced tooth germs were located on the buccal secondary EKs, which correspond to the future paracone in maxilla and protoconid in mandible. The Bmp4 expression in buccal part of the dental mesenchyme might be related with the lower growth in buccal epithelium than in lingual epithelium, and the Msx2 expressing area in epithelium was overlapped with the enamel cord (or septum) and cell dense area. The enamel cord might connect the primary EK with enamel navel to fix the location of the primary EK in the buccal side during the cap to bell stages. Overall, these results suggest that primary EK cells strictly contribute to form the paracone or protoconid, which are the main cusps of the tooth in the maxilla or mandible. [ABSTRACT FROM AUTHOR]

The enamel knot (EK), which is a transient signaling center in the tooth germ, regulates both the differential growth of the dental epithelium and the tooth shape. In this study, the regeneration of the EK was evaluated. The EK regions were removed from the E14 and E16 dental epithelia, and the remaining epithelia were recombined with their original dental mesenchymes. All these tooth germs could develop into calcified teeth after being transplanted into the kidney capsule for 3 weeks. One primary EK was regenerated earlier, and two or three secondary EKs were regenerated later in culture. When simply recombined without removing the EK, the tooth germ, which had four secondary EKs and four cuspal areas of the dental papilla, generated one primary EK first and subsequent secondary EKs. These results indicate that the patterning of the EK in all tooth germs always starts from a primary EK independent of the direct epithelial or mesenchymal control. This suggests that neither the dental epithelium nor the dental mesenchyme can dictate the pattern or number of the EK formation, but the interaction between the dental epithelium and the dental mesenchyme is essential for the regeneration and patterning of the EKs. [ABSTRACT FROM AUTHOR]

Summary: Reciprocal epithelial–mesenchymal interactions control odontogenesis and the cap stage tooth germ mesenchyme specifies crown morphogenesis. The aim of this work was to determine whether this mesenchyme could also control epithelial histogenesis. Dental mesenchyme and enamel organ were dissociated from mouse first lower molars at E14. At this early cap stage, the enamel organ consists of four cell types forming the inner dental epithelium (IDE), primary enamel knot (PEK), outer dental epithelium (ODE) and the stellate reticulum (SR). Pelleted trypsin-dissociated single dental epithelial cells, which had lost all positional information, were reassociated to either dental mesenchyme or dissociated mesenchymal cells and cultured in vitro. Although with different timings, teeth developed in both types of experiments showing a characteristic dental epithelial histogenesis, cusp formation, and the differentiation of functional odontoblasts and ameloblasts. The rapid progression of the initial steps of histogenesis suggested that the cell history was not memorized. The dental mesenchyme, as well as dissociated mesenchymal cells, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. However, the proportion of well-formed multicusped teeth was much higher when intact mesenchyme was used instead of dissociated mesenchymal cells. The mesenchymal cell dissociation had consequences for the functionality of the newly-formed PEK. [Copyright &y& Elsevier]

Runx2 (Cbfa1) is a runt domain transcription factor that is essential for bone development and tooth morphogenesis. Teeth form as ectodermal appendages and their development is regulated by interactions between the epithelium and mesenchyme. We have shown previously that Runx2 is expressed in the dental mesenchyme and regulated by FGF signals from the epithelium, and that tooth development arrests at late bud stage in Runx2 knockout mice [Development 126 (1999) 2911]. In the present study, we have continued to clarify the role of Runx2 in tooth development and searched for downstream targets of Runx2 by extensive in situ hybridization analysis. The expression of Fgf3 was downregulated in the mesenchyme of Runx2 mutant teeth. FGF-soaked beads failed to induce Fgf3 expression in Runx2 mutant dental mesenchyme whereas in wild-type mesenchyme they induced Fgf3 in all explants indicating a requirement of Runx2 for transduction of FGF signals. Fgf3 was absent also in cultured Runx2-/- calvarial cells and it was induced by overexpression of Runx2. Furthermore, Runx2 was downregulated in Msx1 mutant tooth germs, indicating that it functions in the dental mesenchyme between Msx1 and Fgf3. Shh expression was absent from the epithelial enamel knot in lower molars of Runx2 mutant and reduced in upper molars. However, other enamel knot marker genes were expressed normally in mutant upper molars, while reduced or missing in lower molars. These differences between mutant upper and lower molars may be explained by the substitution of Runx2 function by Runx3, another member of the runt gene family that was upregulated in upper but not lower molars of Runx2 mutants. Shh expression in mutant enamel knots was not rescued by FGFs in vitro, indicating that in addition to Fgf3, Runx2 regulates other mesenchymal genes required for early tooth morphogenesis. Also, exogenous FGF and SHH did not rescue the morphogenesis of Runx2 mutant molars. We conclude that Runx2 mediates the functions of epithelial FGF signals regulating Fgf3 expression in the dental mesenchyme and that Fgf3 may be a direct target gene of Runx2. [Copyright &y& Elsevier]

The terminal differentiation of odontoblasts is controlled by the inner dental epithelium (IDE) and occurs according to a tooth-specific pattern. It requires temporospatially regulated epigenetic signaling and the expression of specific competence. The patterning of cusp formation was compared with that of odontoblast differentiation in the first lower molar in mice. Histology, immunostaining, and three dimensional reconstructions were completed by experimental approaches in vitro. The mesenchyme controls the pattern of cusp formation. During the cap-bell transition in the molar, a subpopulation of nondividing IDE cells from the enamel knot (EK) undergo a tooth-specific segregation in as many subpopulations as cusps will form. Epithelial cell-basement membrane interactions seem to be involved in the segregation of EK cells. The timing and spatial pattern of the segregation of EK cells correlate with cusps formation. However, the temporal pattern of odontoblast terminal differentiation is different. This discrepancy might result from cusp-specific differences either in the timing of the initiation of odontoblast terminal differentiation and/or in cell proliferation kinetics. [ABSTRACT FROM AUTHOR]

Teeth develop as epithelial appendages, and their morphogenesis is regulated by epithelial–mesenchymal interactions and conserved signaling pathways common to many developmental processes. A key event during tooth morphogenesis is the transition from bud to cap stage when the epithelial bud is divided into specific compartments distinguished by morphology as well as gene expression patterns. The enamel knot, a signaling center, forms and regulates the shape and size of the tooth. Mesenchymal signals are necessary for epithelial patterning and for the formation and maintenance of the epithelial compartments. We studied the expression of Notch pathway molecules during the bud-to-cap stage transition of the developing mouse tooth. Lunatic fringe expression was restricted to the epithelium, where it formed a boundary flanking the enamel knot. The Lunatic fringe expression domains overlapped only partly with the expression of Notch1 and Notch2, which were coexpressed with Hes1. We examined the regulation of Lunatic fringe and Hes1 in cultured explants of dental epithelium. The expression of Lunatic fringe and Hes1 depended on mesenchymal signals and both were positively regulated by FGF-10. BMP-4 antagonized the stimulatory effect of FGF-10 on Lunatic fringe expression but had a synergistic effect with FGF-10 on Hes1 expression. Recombinant Lunatic fringe protein induced Hes1 expression in the dental epithelium, suggesting that Lunatic fringe can act also extracellularly. Lunatic fringe mutant mice did not reveal tooth abnormalities, and no changes were observed in the expression patterns of other Fringe genes. We conclude that Lunatic fringe may play a role in boundary formation of the enamel knot and that Notch-signaling in the dental epithelium is regulated by mesenchymal FGFs and BMP. [Copyright &y& Elsevier]

Relationships between cell-cell/cell-matrix interactions and enamel organ histomorphogenesis were examined by immunostaining and electron microscopy. During the cap-bell transition in the mouse molar, laminin-5 (LN5) disappeared from the basement membrane (BM) associated with the inner dental epithelium (IDE), and nondividing IDE cells from the enamel knot (EK) underwent a tooth-specific segregation in as many subpopulations as cusps develop. In the incisor, the basement membrane (BM) in contact with EK cells showed strong staining for LN5 and integrin α6β4. LN5 seems to provide stable adhesion, while its proteolytic processing might facilitate cell segregation. In both teeth, immunostaining for antigens associated with desmosomes or adherens junctions was similar for EK cells and neighboring IDE cells. Outside the EK, IDE cell-BM interactions changed locally during the initial molar cusp delimitation and on the labial part of the incisor cervical loop. Conversely, cell-cell junctions stabilized the anterior part of the incisor during completion of morphogenesis. Time and space regulation of cell-matrix and cell-cell interactions might thus play complementary roles in allowing plasticity during tooth morphogenesis and stabilization at later stages of epithelial histogenesis. [ABSTRACT FROM AUTHOR]

Odontoblasts differentiate from the cells of the dental papilla, and it has been well-established that their differentiation in developing teeth is induced by the dental epithelium. In experimental studies, no other mesenchymal cells have been shown to have the capacity to differentiate into odontoblasts, indicating that the dental papilla cells have been committed to odontoblast cell lineage during earlier developmental stages. We propose that the advancing differentiation within the odontoblast cell lineage is regulated by sequential epithelial signals. The first epithelial signals from the early oral ectoderm induce the odontogenic potential in the cranial neural crest cells. The next step in the determination of the odontogenic cell lineage is the development of the dental papilla from odontogenic mesenchyme. The formation of the dental papilla starts at the onset of the transition from the bud to the cap stage of tooth morphogenesis, and this is regulated by epithelial signals from the primary enamel knot. The primary enamel knot is a signaling center which forms at the tip of the epithelial tooth bud. It becomes fully developed and morphologically discernible in the cap-stage dental epithelium and expresses at least ten different signaling molecules belonging to the BMP, FGF, Hh, and Wnt families. In molar teeth, secondary enamel knots appear in the enamel epithelium at the sites of the future cusps. They also express several signaling molecules, and their formation precedes the folding and growth of the epithelium. The differentiation of odontoblasts always starts from the tips of the cusps, and therefore, it is conceivable that some of the signals expressed in the enamel knots may act as inducers of odontoblast differentiation. The functions of the different signals in enamel knots are not precisely known. We have shown that FGFs stimulate the proliferation of mesenchymal as well as epithelial cells, and they may also regulate the growth of the cusps. We have proposed that the enamel knot signals also have important roles, together with mesenchymal signals, in regulating the patterning of the cusps and hence the shape of the tooth crown. We suggest that the enamel knots are central regulators of tooth development, since they link cell differentiation to morphogenesis. [ABSTRACT FROM AUTHOR]

Growth factors belonging to the FGF and TGF-β families, together with other secreted factors such as Sonic hedgehog, have been shown to be spatially and temporally regulated during tooth development. Providing evidence of the functions of these molecules has, however, proved difficult. We have developed a novel strategy for investigating the role of secreted molecules in tooth development using soluble forms of membrane bound receptors to sequester ligands. Chimeric fusion proteins of receptor extracellular domains were cloned into the eukaryotic expression vector pIG-1 and transfected into COS cells. Fusion proteins secreted by the COS cells were purified using Protein A Sepharose. A soluble form of the FGF receptor FGF-1IIIc, which preferentially binds FGF-2 and FGF-4, was produced using this technique and added to mouse mandible cultures. Addition of the soluble receptors to E13 cultures resulted in down-regulation of Sonic hedgehog expression in molar enamel knots, consistent with inhibition of FGF-4 signalling. [ABSTRACT FROM AUTHOR]

While the evolutionary history of mammalian tooth shapes is well documented in the fossil record, the developmental basis of their tooth shape evolution is unknown. We investigated the expression patterns of eight developmental regulatory genes in two species of rodents with different molar morphologies (mouse, Mus musculus and sibling vole, Microtus rossiaemeridionalis). The genes Bmp-2, Bmp-4, Fgf-4 and Shh encode signal molecules, Lef-1, Msx-1 and Msx-2, are transcription factors and p21 CIP1/WAF1 participates in the regulation of cell cycle. These genes are all known to be associated with developmental regulation in mouse molars. In this paper we show that the antisense mRNA probes made from mouse cDNA cross-hybridized with vole tissue. The comparisons of gene expression patterns and morphologies suggest that similar molecular cascades are used in the early budding of tooth germs, in the initiation of tooth crown base formation, and in the initiation of each cusp’s development. Furthermore, the co-localization of several genes indicate that epithelial signalling centres function at the three stages of morphogenesis. The earliest signalling centre in the early budding epithelium has not been reported before, but the latter signalling centres, the primary and the secondary enamel knots, have been studied in mouse. The appearance of species-specific tooth shapes was manifested by the regulatory molecules expressed in the secondary enamel knots at the areas of future cusp tips, whilst the mesenchymal gene expression patterns had a buccal bias without similar species-specific associations. [ABSTRACT FROM AUTHOR]

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14 Cre / Pitx2 flox/flox ( Pitx2 cKO ) and Rosa26 CreERT /Pitx2 flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2 + labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2 , Sox2 , Lef1 and Irx1 ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

An important event in organogenesis is the formation of signaling centers, which are clusters of growth factor-secreting cells. In the case of tooth development, sequentially formed signaling centers known as the initiation knot (IK) and the enamel knot (EK) regulate morphogenesis. However, despite the importance of signaling centers, their origin, as well as the fate of the cells composing them, remain open questions. Here, using lineage tracing of distinct epithelial populations, we found that the EK of the mouse incisor is derived de novo from a group of SRY-box 2 ( Sox2 )-expressing cells in the posterior half of the tooth germ. Specifically, EK progenitors are located in the posterior ventral basal layer, as demonstrated by DiI labeling of cells. Lineage tracing the formed EK with Shh CreER , which encodes an inducible Cre recombinase under the control of the Sonic hedgehog promoter, at subsequent developmental stages showed that, once formed, some EK cells in the incisor give rise to differentiated cells, whereas in the molar, EK cells give rise to the buccal secondary EK. This work thus establishes the developmental origin as well as the fate of the EK and reveals two strategies for the emergence of serially formed signaling centers: one through de novo establishment and the other by incorporation of progeny from previously formed signaling centers., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)