Many vertebrates, from squamates to humans, rely on tooth replacement for long-term dietary needs and protection from predators. Different organisms have adapted to this need by either forming stronger teeth or replacing teeth throughout their lifespan (1). Even though teeth are essential for the survival and success of many organisms, tooth renewal has not been studied very extensively. It was previously difficult to understand due to the fact that development occurs postnatally (3).
Leopard Geckos as a Model Organism
Leopard geckos are an ideal organism to use in tooth regeneration studies because they replace lost teeth continuously throughout their lifetime. Typical geckos have close to 100 exposed teeth at birth and continuously replace them every few months (3).
Leopard Gecko Tooth Replacement
Leopard geckos have tooth ‘families’ that form rows in the lizard’s mouth. Within these rows, new generations of teeth form constantly from before hatching until death. The replacement teeth are formed in the successional lamina and develop lingual (towards the tongue) compared to the erupted teeth. Each replacement tooth grows in size until it eventually surfaces from inside of the tissue and displaces the tooth in it’s place. This turnover process usually takes between 3 and 4 months (3).
Justification for Study
The continuous tooth replacement seen in leopard geckos indicates that stem cells may be involved (3).
Early Tooth Formation
Teeth develop from ectodermal cells which grow inward and thicken in response to outside signaling to form the dental lamina. The dental lamina is a band of epithelial cells that later connects teeth to the oral cavity epithelium (4). Sonic hedgehog (Shh) is expressed in early tooth development. It is located both on the oral epithelia of the dental lamina as well as in the enamel organ (2).
Overview of Tooth Development
Tooth Ontogeny (4)
1 – odontogenic band forms
- derived from ectoderm and mesochyme
- band of gene expression marks the position of future teeth rows;
2 – epithelial cells aggregate and mesenchymal condenses
3 – dental lamina forms
- thickening of epithelium forms dental lamina
- forms basis for later tooth attachment as soon as dental lamina begins to grow
4 – bud stage
- teeth bud on the labial side of the marginal dental lamina and the lingual side of the palatal dental lamina
- side of dental lamina that is at an obtuse angle to the oral epithelium always forms teeth
5 – cap/bell stage
- tooth morphogenesis and histodifferentiation occur
- epithelium thickens on labial side of dental lamina
- multilayered enamel organ is formed that later gives rise to ameloblasts and the successional dental lamina
6 – differentiation late bell stage
- crown shape forms
- odontoblasts and ameloblasts differentiate
- secretion of dentin and enamel
7 – eruption
- new tooth breaks out of dental lamina and replaces tooth in its place
Effects of Shh Signaling on Tooth Formation
Shh has proven to be an extremely important gene in early tooth formation. Firstly, expression of Shh is correlated with the location of tooth rows. At the very beginning of dental formation, the odontogenic band secretes the signal Shh and the transcription factor Pitx2. When the dental lamina begins to form, Shh expression in the oral epithelium domain is broken down into discrete foci. Shh and Ptc1 become expressed on the lingual side during bud formation (5).
Studies on Ptc1 expression indicate that Shh likely does not contribute to the formation of the successional lamina, but has many other important signaling functions. In gene knockout studies, leopard geckos showed severe defects in tooth crown formation including a decrease in size. Shh has proven to be essential in epithelial cell proliferation as well as epithelial cell survival (5).
Experimental Procedures and Results
A pulse-chase test was used to find slowly diving cells in the leopard gecko dental lamina.
Figures A-D track the label retention of cells at weeks 0, 4, 9, and 20. There is a definite decrease in overall label retention because the more proliferative cells stop expressing it. Figure E shows that the label retaining cells after 20 weeks are concentrated in the dental lamina region in the lingual layer. Figure F shows the areas with a large number of highly proliferative cells. The label retaining cells are shown in magenta in Figure G in relation to the remainder of the leopard gecko mouth (3).
Summary of Key Genes Involved in Tooth Development
Dkk3, Igfbp5, Lgr5 – markers of adult stem cells in mammals (3)
Shh – expression helps determine tooth row location formation (3)
Pitx2 – transcription factor for Shh (5)
Wnt – pathway that controls proliferation of dental lamina lingual cells (2)
Tcf3 – espressed in dental lamina, associated with possible stem cells (3)
Tcf4 – expressed in successional lamina, associated with proliferative cells (3)
Conclusions – Dental Epithelial Stem Cells
Dental epithelial stem cells located in the dental lamina are likely essential for tooth regeneration, whether replacement occurs only once or continuously. Cells in the successional lamina are more closely related to cell proliferation. Gene involvement is essential to understanding dental formation in other mammals.
Though the location of epithelial stem cells has been determined, the processes that induce stem cells to divide and form new teeth has not yet been uncovered. Another interesting study could compare monophyodonty, diphyodonty, and polyphyodonty. Because stem cells are responsible for tooth proliferation, it would be interesting to look at the different genes and transcription factors that regulate how many times these stem cells divide.
Monophyodonty – having one set of teeth that are not replaced in life
Diphyodonty – having two sets of teeth that are replaced once in life
Polyphyodonty –having multiple sets of teeth that are replaced continuously throughout life
(1)Handrigan, Gregory R., and Joy M. Richman. “A Network of Wnt, Hedgehog and BMP Signaling Pathways Regulates Tooth Replacement in Snakes.” Developmental Biology 348.1 (2010): 130-41.
(2)Handrigan, Gregory R., and Joy M. Richman. “Autocrine and Paracrine Shh Signaling Are Necessary for Tooth Morphogenesis, but Not Tooth Replacement in Snakes and Lizards (Squamata).” Developmental Biology 337.1 (2010): 171-86.
(3)Handrigan, Gregory R., Kelvin J. Leung, and Joy M. Richman. “Identification of Putative Dental Epithelial Stem Cells in a Lizard with Life-long Tooth Replacement.”Development and Stem Cells 137 (2010): 3545-549.
(4)Richman, Joy M., and Gregory R. Handrigan. “Reptilian Tooth Development.” Genesis 49.4 (2011): 247-60. 1 Apr. 2011.
(5)Vidal, N., and S. Hedges. “The Phylogeny of Squamate Reptiles (lizards, Snakes, and Amphisbaenians) Inferred from Nine Nuclear Protein-coding Genes.” Comptes Rendus Biologies (2005): 1000-008. Elsevier. 27 Oct. 2005.
(6)Thesleff, Irma and Tummers, Mark. “Tooth Organogenesis and Regeneration.” StemBook (2008).