Identification of putative dental epithelial stem cells in a lizard with life-long tooth replacement
ABSTRACT Most dentate vertebrates, including humans, replace their teeth and yet the process is poorly understood. Here, we investigate whether dental epithelial stem cells exist in a polyphyodont species, the leopard gecko (Eublepharis macularius). Since the gecko dental epithelium lacks a histologically distinct site for stem cells analogous to the mammalian hair follicle bulge, we performed a pulse-chase experiment on juvenile geckos to identify label-retaining cells (LRCs). We detected LRCs exclusively on the lingual side of the dental lamina, which exhibits low proliferation rates and is not involved in tooth morphogenesis. Lingual LRCs were organized into pockets of high density close to the successional lamina. A subset of the LRCs expresses Lgr5 and other genes that are markers of adult stem cells in mammals. Also similar to mammalian stem cells, the LRCs appear to proliferate in response to gain of function of the canonical Wnt pathway. We suggest that the LRCs in the lingual dental lamina represent a population of stem cells, the immediate descendents of which form the successional lamina and, ultimately, the replacement teeth in the gecko. Furthermore, their location on the non-tooth-forming side of the dental lamina implies that dental stem cells are sequestered from signals that might otherwise induce them to differentiate.
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ABSTRACT: 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.Frontiers in Physiology 10/2014; 5:386. DOI:10.3389/fphys.2014.00386
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ABSTRACT: The Mexican tetra (Astyanax mexicanus), a fresh water teleost fish, is an excellent vertebrate model organism to study tooth development, specifically the spatiotemporal events related to the development of the oral and pharyngeal dentitions. In contrast to the coordinated early tooth development in the premaxilla and mandible, the maxillary teeth develop much later in life at 60 dpf. By analysing a growth series of bone and cartilage stained tetra and histological sectioning of the tooth bearing bones, we track the developmental events of tooth development over ontogeny of this animal. Whole mount in situ hybridization with bone morphogenetic proteins and their inhibitor Noggin was conducted to track the late tooth development events. Our data shows that the first generation teeth are small and unicuspid irrespective of their location. Oral jaw teeth become multicuspid and large over ontogeny while the pharyngeal dentition remains unicuspid and disorganised. Tooth eruption occurs late in the maxillary bone. The distinct expression pattern of the BMP antagonist, Noggin, suggests that Noggin plays an inhibitory role by preventing early tooth development in the maxillary bone. This data further supports and highlights the use of the Mexican tetra in understanding the spatio-temporal differences in tooth development in vertebrate jaws.Mechanisms of Development 10/2014; DOI:10.1016/j.mod.2014.09.002 · 2.24 Impact Factor
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ABSTRACT: Abstract The zebrafish, a model organism for which a plethora of molecular and genetic techniques exists, has a lifelong replacing dentition of 22 pharyngeal teeth. This is in contrast to the mouse, which is the key organism in dental research but whose teeth are never replaced. Employing the zebrafish as the main organism to elucidate the mechanisms of continuous tooth replacement, however, poses at least one major problem, related to the fact that all teeth are located deep inside the body. Investigating tooth replacement thus relies on conventional histological methods, which are often laborious, time-consuming and can cause tissue deformations. In this review, we investigate the advantages and limitations of adapting current visualization techniques to dental research in zebrafish. We discuss techniques for fast sectioning, such as vibratome sectioning and high-resolution episcopic microscopy, and methods for in toto visualization, such as Alizarin red staining, micro-computed tomography, and optical projection tomography. Techniques for in vivo imaging, such as two-photon excitation fluorescence and second harmonic generation microscopy, are also covered. Finally, the possibilities of light sheet microscopy are addressed.Zebrafish 01/2015; 12(1). DOI:10.1089/zeb.2014.0980 · 2.88 Impact Factor