Gas1 Regulates Patterning of the Murine and Human Dentitions through Sonic Hedgehog.

The mammalian dentition is a serially homogeneous structure that exhibits wide numerical and morphological variation among multiple different species. Patterning of the dentition is achieved through complex reiterative molecular signaling interactions that occur throughout the process of odontogenesis. The secreted signaling molecule Sonic hedgehog (Shh) plays a key role in this process, and the Shh coreceptor growth arrest-specific 1 (Gas1) is expressed in odontogenic mesenchyme and epithelium during multiple stages of tooth development. We show that mice engineered with Gas1 loss-of-function mutation have variation in number, morphology, and size of teeth within their molar dentition. Specifically, supernumerary teeth with variable morphology are present mesial to the first molar with high penetrance, while molar teeth are characterized by the presence of both additional and absent cusps, combined with reduced dimensions and exacerbated by the presence of a supernumerary tooth. We demonstrate that the supernumerary tooth in Gas1 mutant mice arises through proliferation and survival of vestigial tooth germs and that Gas1 function in cranial neural crest cells is essential for the regulation of tooth number, acting to restrict Wnt and downstream FGF signaling in odontogenic epithelium through facilitation of Shh signal transduction. Moreover, regulation of tooth number is independent of the additional Hedgehog coreceptors Cdon and Boc, which are also expressed in multiple regions of the developing tooth germ. Interestingly, further reduction of Hedgehog pathway activity in Shhtm6Amc hypomorphic mice leads to fusion of the molar field and reduced prevalence of supernumerary teeth in a Gas1 mutant background. Finally, we demonstrate defective coronal morphology and reduced coronal dimensions in the molar dentition of human subjects identified with pathogenic mutations in GAS1 and SHH/GAS1, suggesting that regulation of Hedgehog signaling through GAS1 is also essential for normal patterning of the human dentition.

The supernumerary cheek tooth in tabby/EDA mice-a reminiscence of the premolar in mouse ancestors.

OBJECTIVE: A supernumerary cheek tooth occurs mesially to the first molar in tabby/EDA (Ta) mice affected by hypohidrotic ectodermal dysplasia. The supernumerary tooth (S) has been hypothetically homologized to the premolar, which has disappeared during mouse evolution. DESIGN: This hypothesis was tested using available morphological data on the lower cheek teeth in wild type (WT) and Ta mice. RESULTS: The presence of S is accompanied by a reduction in the mesial portion of the M(1) in mutant mice. 3D reconstructions suggest that the S in Ta homo/hemizygous embryos originates from a split off the mesial portion of the first molar (M(1)) cap. In WT embryos, two vestigial tooth primordia are transiently distinct in front of the M(1). The distal vestige has the form of a wide bud and participates during the development of the mesial portion of the M(1). This bud has been homologized with the vestigial primordium of the fourth premolar of mouse ancestors. The premolar disappearance coincided with a mesial lengthening of the M(1) during mouse evolution. The incorporation of the distal premolar vestige into the mesial part of the M(1) in WT embryos can be regarded as a repetition of the premolar disappearance during evolution. CONCLUSION: : Ontogenetic and phylogenetic data support that the S in Ta mice arises due to the segregation of the distal premolar vestige from the molar dentition and thus represents an evolutionary throwback (atavism).

Development of the vestigial tooth primordia as part of mouse odontogenesis.

The mouse functional dentition comprises one incisor separated from three molars by a toothless diastema in each dental quadrant. Between the incisor and molars, the embryonic tooth pattern also includes vestigial dental primordia, which undergo regression involving apoptosis in their epithelium. Apoptosis appears to play an important role in achieving the specific tooth pattern in the mouse. We documented similarities in the folding mechanism allowing the formation of the dental lamina in mice as well as in reptiles. While further budding on this dental lamina gives rise to many individual simple tooth primordia in crocodiles and lizards, budding morphogenesis of several simple tooth primordia appears to be integrated in the mouse, giving rise to enamel organs of a complex nature. The differentiation of a mammalian tooth germ during both ontogeny and phylogeny might thus include the concrescence (connation) of more primordia, putatively corresponding to simple teeth in mammalian ancestors.

The presence of rudimentary odontogenic structures in the mouse embryonic mandible requires reinterpretation of developmental control of first lower molar histomorphogenesis.

In the mouse embryonic maxilla, rudimentary tooth primordia have been identified, which can be mistaken for the first upper molar. In order to determine whether such a situation might exist in the lower jaw as well, tooth development was investigated in the mouse mandibular cheek region during ED 12.5-15.0. A combination of histology, morphometry and computer-aided 3D reconstructions demonstrated the existence of rudimentary dental structures, whose gradual appearance and regression was associated with the segmental progress of odontogenesis along the mesio-distal axis of the jaw: 1) At ED 12.5, the mesial segment (MS) was the most prominent part of the dental epithelial invagination. It included an asymmetrically budding dental lamina. The MS, although generally mistaken for the lower first molar (M1, primordium, regressed and did not finally participate in M1 cap formation. 2) At ED 13.5, a wide dental bud (called segment R2) appeared distally to the MS. Although the R2 segment transiently represented the predominant part of the dental epithelium at ED13.5, it participated only in the formation of the mesial end of the M1 cap. 3) The top of the R2 segment at ED13.5 was not the precursor of the enamel knot (EK), contrary to what has been assumed. 4) The central segment of the M1 cap as well as the EK developed later and distally to the R2 segment. 5) Time-space specific apoptosis correlated with the retardation in growth of the R2 segment as well as with strong regressive changes in the epithelium situated mesially to it. These highlight the need to reinterpret current molecular data on early M1 development in the mouse in order to correlate the expression of signalling molecules with specific morphogenetic events in the appropriate antemolar or molar segments of the embryonic mandible.

Dentition development and budding morphogenesis.

The development of functional teeth in the mouse has been widely used as a model to study general mechanisms of organogenesis. Compared with other mammals, in which three incisors, one canine, four premolars, and three molars may occur even in each dental quadrant, the mouse functional dentition is strongly reduced. It comprises only one incisor separated from three molars by a toothless gap diastema at the location of the missing teeth. However, mouse embryos also develop transient vestigial dental primordia between the incisor and molar germs in both the upper and lower jaws. These rudimental structures regress, and epithelial apoptosis is involved in this process. The existence of the vestigial dental structures allowed a better assessment of the periodicity in the mouse dentition, which extends opportunities for the interpretation of molecular data on tooth development. We compared the dentition development with tentative models of budding morphogenesis in other epithelial appendages lungs and feathers. We suggested how developmental control by signaling molecules, including bone morphogenetic protein (Bmp), sonic hedgehog (Shh), and fibroblast growth factor (Fgf), can be similarly involved during budding morphogenesis of dentition and other epithelial appendages. We propose that epithelial apoptosis plays an important role in achieving specific features of dentition, whose development involves both budding and its more complex variant branching. The failure of segregation of the originating buds supports the participation of the concrescence of several tooth primordia in the evolutionary differentiation of mammalian teeth.

Karyological and dental identification of Microtus limnophilus in a large focus of alveolar echinococcosis (Gansu, China).

A study of voles (Arvicolidae, Rodentia) from Gansu (China) designed to identify a potential host of Echinococcus multilocularis, responsible for human alveolar echinococcosis, leads to a general analysis of Microtus limnophilus population karyotypes, M1 of M. oeconomus populations from all of Eurasia and of M. limnophilus of Mongolia. The Microtus of Gansu belonging to the nominal subspecies M. limnophilus limnophilus (2n = 38; NF = 58) differs markedly in size and shape of M1 from the M. limnophilus of Mongolia, which must therefore be considered as a new subspecies M. limnophilus of malygini nov. ssp. (2n = 38; NF = 60) and the M. oeconomus of Mongolia should be ranked as M. oeconomus kharanurensis nov. ssp. (2n = 30; NF = 60).

Initial features of the inner dental epithelium histo-morphogenesis in the first lower molar in mouse.

First lower molar development in the mouse was investigated from the cap to early bell stage using histology, morphometry, TEM and 3D reconstructions. This period was characterized by the histogenesis of the enamel organ (EO), folding of the epithelio-mesenchymal junction and growth of the tooth. The histogenesis of the EO and appearance of the enamel knot (EK) were initiated at the early cap stage (ED14). From ED14 to ED15, the anterior and posterior extension of the EK was very prominent whilst the length of the enamel organ did not substantially change. The EK appeared as a dynamic and transitory histological structure including dying and replacement cells. At ED16, the folding of the IDE, which extended over the anterior two thirds of the molar, was the first sign of cuspidogenesis. It was accompanied by a local remodeling of the basement membrane (BM): IDE cells involved in this folding transitorily lost contact with the BM which formed a loop in the mesenchyme. During this period, the growth of the lower M1 along the antero-posterior axis was restricted to the posterior part of the molar. Histogenesis occurred in the whole EO, whilst initial cuspidogenesis was limited to the anterior part of the tooth. Distinct cell populations were thus involved in different contemporary processes leading to changes in the cell density in the mesenchyme, in the mitotic activity, in cell-shape, and cell-matrix interactions in the IDE, and remodeling of the BM where both epithelium and mesenchyme might participate.

Correlation between apoptosis distribution and BMP-2 and BMP-4 expression in vestigial tooth primordia in mice.

The eutherian dental formula consists of three incisors, one canine, four premolars and three molars in each dental quadrant. Muroid evolution led to a reduction in the number of teeth, with one incisor separated from three molars by a long diastema. However, seven vestigial tooth primordia (D1-5, R1-2) have been detected in the embryonic diastemal area of the mouse maxilla between embryonic days (ED) 12.5 and 13.5. Computer-aided 3D reconstructions were used to analyse the temporo-spatial pattern of apoptosis during regression in the two largest and most distal vestiges (R1, R2). These structures have been widely considered as the primordium of the first upper molar and, accordingly, related molecular data have been interpreted exclusively in terms of progressive molar development. The spatial distribution of epithelial apoptosis, which affected the R1 and R2 rudiments in two consecutive waves on ED 12.5 and 13.5, respectively, was compared with our earlier data on expression of genes encoding bone morphogenetic proteins (BMP-2 and BMP-4). Similar temporo-spatial patterns of apoptosis and expression of BMP, specifically confined to the epithelium of the rudimentary tooth primordia, strongly support involvement of BMPs in the regulation of epithelial apoptosis during odontogenesis.

Early stages of tooth morphogenesis in mouse analyzed by 3D reconstructions.

Computer-aided 3D reconstructions were used to investigate early odontogenesis in the ICR mouse, from the dental lamina to the cap stage. The diastemal region of the maxilla was not an empty zone: five transient epithelial rudiments (D1-D5) were found between ED 12.5-13.5. Two further rudiments (R1 and R2) were observed between D5 and the maxillary first molar primordium, whose bud emerged at ED 13.5. These rudiments might be related to vestiges of ancestral teeth. During this period, only an epithelial lamina was observed in front of the bud-shaped molar epithelium in the cheek region of the mandible. Apoptosis plays an important role in the reduction of antemolar rudiments in the maxilla and in the remodeling of the epithelium anterior to the M1 bud and cap in both jaws: two successive waves of apoptosis were detected in the mandible and in the maxilla. Computer-aided 3D reconstructions clearly demonstrated that morphologically different developmental stages coexist along the anteroposterior axis of M1 in both jaws.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. III. Spatial distribution of mitoses and apoptoses up to bell-staged first lower molar teeth.

Computer-assisted 3D reconstructions were used to follow the development of the embryonic mouse first lower molar (M1). At ED 12.5, the thickening of the oral epithelium, which was thought to correspond to the molar dental lamina, regressed in its anterior part as a result of apoptosis. Only the posterior part later gave rise to molars. The transition to the cap stage entailed medial and lateral extensions of the dental epithelium. The growth and histo-morphogenesis of the enamel organ as well as cervical loop formation proceeded more rapidly in the anterior part of the M1 during the cap and early bell stages producing significant morphological differences along the antero-posterior axis. Apoptosis was temporarily intensive in the anterior part of the bud- and cap-shaped epithelium and thus pointed domains which do not participate in the formation of the final M1 enamel organ. In the well-formed cap, apoptoses displayed maximum concentration in the enamel knot (EK). No increase in the number of metaphases could be detected in the vicinity of the EK. Mitoses were distributed throughout the epithelial compartment until cap stage and then mainly concentrated in the inner dental epithelium at the early bell stage. At this later stage, either lateral views or thick virtual sections performed in the reconstruction demonstrated a clear cut distribution of mitoses and apoptoses in the enamel organ. At the early bell stage, mitoses in the mesenchyme demonstrated an increasing postero-anterior gradient.