Egg Tooth Morphogenesis in the Leopard Gecko: A Model for Dental Size Variation

Tooth size can vary along a tooth row in an individual organism, and can also change through time as new teeth replace erupted ones. Although much is known about morphoregulation of the dentition in mammals, little is known about what controls tooth size and shape in other heterodont amniotes. In this study, we examine tooth development through growth in the leopard gecko Eublepharis macularis in order to understand the timing of tooth mineralization and its contribution to sizing differences in teeth. The embryonic leopard gecko has greatly enlarged, anteriorly oriented egg teeth that are shed a few days after hatching, and show a dramatic phenotype for studying size variation in an organ. Typical tooth replacement in polyphyodont animals involves the replacement of a small tooth with a larger tooth through growth, whereas in the hatchling leopard gecko several smaller teeth replace the very large egg tooth. In this study, we aimed to: 1) quantify differences in tooth size at the very earliest stages of tooth development; and 2) visualize the mineralization pattern in egg and marginal teeth in ovo. An understanding of the timing and process of tooth mineralization in the leopard gecko will serve as a framework to address larger questions of development and the control of shape and size of teeth, especially when considering variation in heterodont dentitions in polyphyodont animals. To document the development of the egg tooth and marginal teeth, 13 specimens were fixed at different developmental stages: 6 weeks before hatching to hatching, before the egg teeth were shed. Specimens were examined histologically with and without decalcification, and cells in developing tooth buds were counted as a proxy for the size of the tooth germ. In separate experiments, embryos were injected with two fluorescent dyes, calcein and xylenol orange, 5 days apart in ovo to mark teeth that were actively mineralizing. The entire tooth row was examined with Optical Projection Tomography (OPT) to view the fluorescent mineralization fronts in 3D. Subsequently, specimens were stained with phosphotungstic acid to enhance contrast and micro CT scanned to observe the morphology of the dental lamina and egg teeth at hatching. Results show that at stage 33 (~14 days post oviposition) the egg tooth is at the cap stage, whereas the rest of the functional teeth are at bud stage or earlier. The dental placode for the egg tooth has a higher cell count than those of the marginal teeth. The fluorescent mineralization fronts indicate that the egg tooth remains in the histogenesis phase for a longer period of time than the marginal teeth, and that mineralization is asymmetrical in the last stages of embryonic growth to create the forward curvature of the tooth. The increased size of the egg tooth is due in part to early initiation and a greater number of cells allocated to the dental placode. Further experiments will determine whether other mechanisms such as increased proliferation and accelerated differentiation are involved in regulating tooth organ size. Support or Funding Information Michael Smith Foundation for Health Research, Killam Postdoctoral Fellowship, National Institutes of Health, National Sciences and Engineering Research Council of Canada