Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling.

Much of our knowledge about mammalian evolution comes from examination of dental fossils, because the highly calcified enamel that covers teeth causes them to be among the best-preserved organs. As mammals entered new ecological niches, many changes in tooth number occurred, presumably as adaptations to new diets. For example, in contrast to humans, who have two incisors in each dental quadrant, rodents only have one incisor per quadrant. The rodent incisor, because of its unusual morphogenesis and remarkable stem cell-based continuous growth, presents a quandary for evolutionary biologists, as its origin in the fossil record is difficult to trace, and the genetic regulation of incisor number remains a largely open question. Here, we studied a series of mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor-tyrosine kinase signaling. In sprouty loss-of-function mutants, splitting of gene expression domains and reduced apoptosis was associated with subdivision of the incisor primordium and a multiplication of its stem cell-containing regions. Interestingly, changes in sprouty gene dosage led to a graded change in incisor number, with progressive decreases in sprouty dosage leading to increasing numbers of teeth. Moreover, the independent development of two incisors in mutants with large decreases in sprouty dosage mimicked the likely condition of rodent ancestors. Together, our findings indicate that altering genetic dosage of an antagonist can recapitulate ancestral dental characters, and that tooth number can be progressively regulated by changing levels of activity of a single signal transduction pathway.

The dynamics of supernumerary tooth development are differentially regulated by Sprouty genes.

In mice, a toothless diastema separates the single incisor from the three molars in each dental quadrant. In the prospective diastema of the embryo, small rudimentary buds are found that are presumed to be rudiments of suppressed teeth. A supernumerary tooth occurs in the diastema of adult mice carrying mutations in either Spry2 or Spry4. In the case of Spry2 mutants, the origin of the supernumerary tooth involves the revitalization of a rudimentary tooth bud (called R2), whereas its origin in the Spry4 mutants is not known. In addition to R2, another rudimentary primordium (called MS) arises more anteriorly in the prospective diastema. We investigated the participation of both rudiments (MS and R2) in supernumerary tooth development in Spry2 and Spry4 mutants by comparing morphogenesis, proliferation, apoptosis, size and Shh expression in the dental epithelium of MS and R2 rudiments. Increased proliferation and decreased apoptosis were found in MS and R2 at embryonic day (ED) 12.5 and 13.5 in Spry2(-/-) embryos. Apoptosis was also decreased in both rudiments in Spry4(-/-) embryos, but the proliferation was lower (similar to WT mice), and supernumerary tooth development was accelerated, exhibiting a cap stage by ED13.5. Compared to Spry2(-/-) mice, a high number of Spry4(-/-) supernumerary tooth primordia degenerated after ED13.5, resulting in a low percentage of supernumerary teeth in adults. We propose that Sprouty genes were implicated during evolution in reduction of the cheek teeth in Muridae, and their deletion can reveal ancestral stages of murine dental evolution.

Patterning by heritage in mouse molar row development.

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the "primary enamel knot" (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

Shh expression in a rudimentary tooth offers new insights into development of the mouse incisor.

For teeth as for any organ, knowledge of normal development is essential for the proper interpretation of developmental anomalies in mutant mice. It is generally accepted that tooth formation is initiated with a single signaling center that, in the incisor region, is exclusively related to the development of the functional adult incisor. Here, using a unique combination of computer-aided three-dimensional reconstructions and whole mount in situ hybridization of mandibles from finely staged wild-type mouse embryos, we demonstrate that several Sonic hedgehog (Shh) expression domains sequentially appear in the lower incisor region during early development. In contrast to the single Shh expression domain that is widely assumed to be present in each lower incisor area at ED12.5-13.5, we identified two spatially distinct regions of Shh expression that appear in an anterior-posterior sequence during this period. The initial anterior, more superficially located Shh expression region represented the rudimentary (so-called deciduous) incisor, whereas only the later posterior deeper situated region corresponded to the prospective functional incisor. In the more advanced embryos, only this posterior Shh expression in the incisor bud was detectable as a precursor of the enamel knot. This study offers a new interpretation of published molecular data on the mouse incisor from initiation through ED13.5. We suggest that, as with Shh expression, other molecular data that have been ascribed to the progressive development of the mouse functional incisor at early stages, in fact, correspond to a rudimentary incisor whose development is aborted.

Revitalization of a diastemal tooth primordium in Spry2 null mice results from increased proliferation and decreased apoptosis.

An understanding of the factors that promote or inhibit tooth development is essential for designing biological tooth replacements. The embryonic mouse dentition provides an ideal system for studying such factors because it consists of two types of tooth primordia. One type of primordium will go on to form a functional tooth, whereas the other initiates development but arrests at or before the bud stage. This developmental arrest contributes to the formation of the toothless mouse diastema. It is accompanied by the apoptosis of the rudimentary diastemal buds, which presumably results from the insufficient activity of anti-apoptotic signals such as fibroblast growth factors (FGFs). We have previously shown that the arrest of a rudimentary tooth bud can be rescued by inactivating Spry2, an antagonist of FGF signaling. Here, we studied the role of the epithelial cell death and proliferation in this process by comparing the development of a rudimentary diastemal tooth bud (R(2)) and the first molar in the mandibles of Spry2(-/-) and wild-type (WT) embryos using histological sections, image analysis and 3D reconstructions. In the WT R(2) at embryonic day 13.5, significantly increased apoptosis and decreased proliferation were found compared with the first molar. In contrast, increased levels of FGF signaling in Spry2(-/-) embryos led to significantly decreased apoptosis and increased proliferation in the R(2) bud. Consequently, the R(2) was involved in the formation of a supernumerary tooth primordium. Studies of the revitalization of rudimentary tooth primordia in mutant mice can help to lay the foundation for tooth regeneration by enhancing our knowledge of mechanisms that regulate tooth formation.