Many reptiles are able to continuously replace their teeth through life, an ability attributed to the existence of epithelial stem cells. Tooth replacement occurs in a spatially and temporally regulated manner, suggesting the involvement of diffusible factors, potentially over long distances. Here, we locally disrupted tooth replacement in the leopard gecko (Eublepharis macularius) and followed the recovery of the dentition. We looked at the effects on local patterning and functionally tested whether putative epithelial stem cells can give rise to multiple cell types in the enamel organs of new teeth. Second generation teeth with enamel and dentine were removed from adult geckos. The dental lamina was either left intact or disrupted in order to interfere with local patterning cues. The dentition began to reform by 1 month and was nearly recovered by 2–3 months as shown in μCT scans and eruption of teeth labeled with fluorescent markers. Microscopic analysis showed that the dental lamina was fully healed by 1 month. The deepest parts of the dental lamina retained odontogenic identity as shown by PITX2 staining. A pulse-chase was carried out to label cells that were stimulated to enter the cell cycle and then would carry BrdU forward into subsequent tooth generations. Initially we labeled 70–78% of PCNA cells with BrdU. After a 1-month chase, the percentage of BrdU + PCNA labeled cells in the dental lamina had dropped to 10%, consistent with the dilution of the label. There was also a population of single, BrdU-labeled cells present up to 2 months post surgery. These BrdU-labeled cells were almost entirely located in the dental lamina and were the likely progenitor/stem cells because they had not entered the cell cycle. In contrast fragmented BrdU was seen in the PCNA-positive, proliferating enamel organs. Homeostasis and recovery of the gecko dentition was therefore mediated by a stable population of epithelial stem cells in the dental lamina.

Tooth replacement is a conserved process in vertebrates extending back to the ancesters of modern fish, amphibians, reptiles and mammals. The patterns in dinosaurs and reptiles have been described by scientists as regular waves going from posterior to anterior of the jaws where every other tooth is replaced. The patterns suggest that local and possibly jaw-wide communication betwen tooth families is involved. The tooth replacement process is not well understood since there are few animals with acessible dentitions. In this study we study leopard geckos in which tooth succession occurs repeatedly throughout life thus giving multiple opportunities to track the process. In addition we performed 2 types of experiments that unlaterally interfere with tooth replacement. Animals were followed using serial wax bites for 2-3 months prior to unilateral, selective tooth removal and then for 7-9 months after surgery. The data on tooth presence or absence was transformed mathematically to capture the time between eruption events of the successional tooth at the same position. Relationships between neighbouring tooth families and across the midline were measured to look for relative phase symmetry or asymmetry. We then tested 5 alternative models of tooth replacement to explain the observed patterns of recovery. The selective removal of unerupted second generation teeth showed that there were no signals passing back to the tooth forming field. The pattern of tooth eruption was not only re-established but it was in phase with the surrounding unperturbed tooth locations, as reflected in the nearest-neighbour diagonal lines. In animals that had permanent ablation of the dental lamina, the cycling of teeth anterior and posterior to the gap was unaffected refuting the presence of directional factors that pass from one tooth family to the next. Finally we compared the patterns of replacement between geckos and alligators using Edmund, 1962 data. The general nearest neighbour staggered patterns were conserved but the alligator had very slower rates of replacement in the posterior jaws. The geckos maintain similar cycling frequences across the jaws. In conclusion, we reject the Osborn model of zones of local inhibition and Edmunds wave-stimulus theory.Instead propose that teeth themselves are cycling and that these cycles regulate the staggered timing of tooth initiation. We also showed that once established, the local control of tooth replacement is very resilient to environmental perturbations as long as the dental epithelium is retained.

The aim of this study is to profile the transcriptomes of teeth and the surrounding tissues of an adult lizard dentition (Eublepharis macularius) that is actively replacing teeth throughout life. Bulk RNAseq was used to compare teeth that are in function versus unerupted, developing teeth and single cell RNA-seq was carried out on jaw segments containing the dental forming tissues. In bulk RNAseq data, we found that functional teeth expressed genes involved in bone and tooth resorption. Indeed, multinucleated odontoclasts were abundant in tissue sections of functional teeth undergoing resorption. These are the first steps towards characterizing odontoclasts that participate in physiological resorption. Unexpectedly, chemotaxis gene SEMA3A was expressed within odontoblasts and in adjacent mesenchyme, confirmed using RNAscope. Semaphorins may be involved in regulating odontoclasts during tooth resorption. The scRNA-seq experiment successfully isolated dental mesenchyme and epithelial cells. We confirmed that some of these genes are expressed in the earliest tooth buds within the tooth forming field. In addition, we found evidence of convergent evolution in the tooth eruption trait. Geckos evolved a means for second generation teeth to communicate with the functional teeth. Instead of a dental follicle inducing an eruption pathway as in the mammal, the gecko and other squamate reptiles use the enamel organ of the successional teeth to trigger tooth resorption of the functional teeth, thus creating an eruption pathway. New molecules such as SEMA3A may also participate in this process. Future studies on the gecko will uncover the molecular basis of convergent evolution in the dentition.

Introduction and Objectives: The ability to replace teeth throughout life is a trait found in most vertebrates and was present in ancient mammals. Modern mammals including humans can only replace their teeth once and much effort is currently being devoted to uncovering stem cells to be used in tooth regeneration. Work from our lab and others has identified some of the differences in the reptilian dentition that could underlie the basis of polyphyodonty. In lizards, the dental lamina, a source of putative dental epithelial progenitor cells, persists until adulthood. In contrast, in humans there are very few remnants of the dental lamina remaining once the permanent dentition is complete. In this project we use the leopard gecko (Eublepharis macularius) and surgical treatments to trace the contribution of label‐retaining cells to the next generation of teeth. We hypothesized that premature removal of unerupted second generation teeth will stimulate populations of quiescent label‐retaining cells to proliferate. Then by following the labeled cells for several months we will identify progeny of the epithelial progenitor cells in newly formed teeth. Materials and Methods: Second generation, unerupted teeth were removed from geckos under sedation. A pulse of BrdU was administered over 3 sequential days. Animals were euthanized at 1 week post surgery (immediately after the BrdU pulse or time 0) and 1 or 2 months post surgery (N = 3 animals per timepoint). Animals were processed for immunofluorescence staining with BrdU antibodies. We counted the proportion of nuclei in the dental lamina that stained positive for BrdU. Results: One week after the surgery, we identified statistically significant higher uptake of BrdU in the dental epithelium of treated as compared to the control, non‐manipulated regions (P < 0.01). One month after the surgery, there was a significant decrease in BrdU label in the dental lamina (P< 0.001), decreasing to the same level as controls at time zero The newly formed tooth buds regenerating in the sites of tooth removal contained BrdU labeled cells in all parts of the enamel organ. The original label‐retaining cells divided several times since the original pulse, based on the fragmented appearance of the BrdU in the nuclei. Conclusion: In our previous work, it was difficult to label the quiescent cells of the dental lamina. Here the removal of the successional teeth stimulates proliferation of the dental lamina. With a larger population of label‐retaining cells that could be tracked we were able to find transit amplifying cells that divide and contribute to the enamel organ. Significance: We have developed an injury model that will allow us to test the ability of different signals to regulate the activity of stem cells. Moreover label‐retaining cells are multipotent and can self‐renew, thereby possessing some of the properties of progenitor cells.