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.

The Olive Ridley (Lepidochelys olivacea), is the most abundant species of sea turtles in the world, but is still threatened according to the red list of endangered species. Apparently, the Olive Ridley turtle seems more prone to congenital malformations compared to other species of sea turtles. In northwestern Mexico, an incidence of congenital malformations of 2% has been reported for this species, most of which occur in the craniofacial region (49%). The objective of this study was to deduce the etiology of the malformations based on the pattern of the affected structures. During the 2019 nesting season at El Caimanero nesting beach, Sinaloa, Mexico, 11 specimens with external craniofacial malformations and 10 that did not show external malformation were collected. The normal animals hatched, but died from other natural causes within the nests. Specimens were divided between histological analysis or µCT with phosphotungstic acid contrast enhancement. Specimens with malformations consistent with a Holoprosencephaly (HPE) phenotype were detected (Table 1). Analysis PTA staining and µCT showed there was a single external naris. There was variability in the morphology of the nasal passages. Other midline structures were also deficient including absent interorbital septum and small premaxilla. However, two other cardinal signs of severe HPE were not seen. The brain was divided into right and left hemispheres and the eyes were separated. There were additional defects in the mandible affecting mesodermal derivatives of the first arch. The tongue was severely hypoplastic and the musculature in the floor of the oral cavity was deficient. The tracheal opening was also displaced posteriorly in the tongue compared to the usual position in the center of the tongue. The nasal defects may be caused by a hypoplastic frontonasal process which allows merging of the nasal pits. The muscular deficiencies in the tongue are likely due to incomplete formation of lingual swellings in the first pharyngeal arch. Nasal development was normal in the majority of animals and the most common defect was mandibular deviation combined with hypoplasia. The defects observed are consistent with a mild form of HPE but also with ciliopathies that include tongue agenesis. Further histological analysis will be carried out to assess cellular and molecular phenotypes. This work suggest pleotropic environmental effects cause the embryonic defects after the eggs are oviposited in the nest. This turtle species lays eggs with embryos in the gastrula stage of development, reaching pharyngula stage, 3‐4 days after laying. Therefore, we postulate an exposure to an environmental teratogen in the first 4 days is responsible for the congenital abnormalities.