Fluorescent Labeling in the Leopard Gecko Resolves Tooth Initiation Patterns in ovo and Suggests Origins of Post‐hatching Shedding Patterns

The unerupted teeth of the polyphyodont leopard gecko form a highly patterned array consisting of teeth arranged in diagonal lines from most to least mature. These gradients of tooth maturity and size were originally termed Zahnreihen. Little is known about how these arrays assemble in such a precise pattern. We therefore set out to follow the addition of teeth to the Zahnreihen in geckos. First we scanned 5 hatchlings with μCT to quantify the number and arrangement of teeth specified in ovo. All individuals had diagonally oriented Zahnreihen consisting of 4–5 teeth along the jaw arch. Embryos were fixed at several times prior to hatching to investigate the development of the Zahnreihen. The first teeth to mineralize in the maxilla become the anterior‐most members of the longest Zahnreihen (5 teeth long). Marginal teeth are added anteriorly and posteriorly to this region before the Zahnreihen elongate. Nonetheless, the relative positions of some developing teeth and total marginal tooth numbers differed between individuals at hatching. Contributing to this variability, in between larger teeth at the jaw margin, we identified small, mineralized structures in between larger marginal teeth that could be rudimentary teeth or newly initiating tooth positions (and possibly Zahnreihen). To clarify the identity of these small marginal structures, we performed a dual label experiment with calcein and xylenol orange to document mineralization of teeth in ovo (N = 3). Calcein was injected at stage 39 at a time when μCT scans show 13–14 mineralizing teeth per upper jaw quadrant. Xylenol orange was injected 5 days later, and then embryos were sacrificed just prior to hatching, 15 days after the xylenol injection. 3D imaging with optical projection tomography revealed that the smallest, lingual‐most teeth have only a xylenol label, indicating that the dyes are cleared from embryonic circulation despite being in the closed system of the egg. The small mineralized structures identified in the marginal tooth row were labeled with calcein, indicating that they represent rudimentary teeth initiated early in tooth patterning, not the interstitial addition of new tooth positions peri‐hatching. No unlabeled teeth were present along the jaw, so in the 15 days post‐labeling, no new teeth formed that could be detected within the resolution of our scans (9.55μm voxel size). The proportion of calcein to xylenol label within volume renderings of developing teeth revealed synchronous deposition of matrix across homologous teeth in adjacent Zahnreihen. Rudimentary teeth did not begin a Zahnreihe. Finer details of the labeling reveal small variations in matrix deposition among teeth of similar age. Our lab previously found that post‐hatching shedding patterns (wave replacement and smaller variations within it such as premaxillary/maxillary regionalization and fixed slight asymmetries) are maintained from when this first dentition is shed into later tooth generations. The results of this study suggest that consistent deviations in post‐hatching shedding patterns may come from in ovo patterning directly. Support or Funding Information National Sciences and Engineering Research Council, National Institutes of Health, Michael Smith Foundation for Health Research, Killam postdoctoral fellowship