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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., Competing Interests: TG is currently employed by the company STEMCELL Technologies. All lab work performed by TG was prior to employment at STEMCELL Technologies. STEMCELL Technologies has no financial or experimental contribution to this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Brink, Henríquez, Grieco, Martin del Campo, Fu and Richman.)

The canonical (β-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts.