Understanding the mechanisms that control tooth replacement

As a child losing a tooth is a cause for celebration with a visit from the tooth fairy. Later in life, tooth loss becomes a significant issue, with the prospect of implants, bridges, and dentures. This is because as mammals we only have two sets of teeth, our baby (deciduous) teeth, and our permanent teeth. This brings the question of why we have this restriction when elsewhere in the animal kingdom sharks, snakes and crocodiles have a seemingly unlimited supply of replacement teeth. Here we aim to understand the mechanisms that restrict tooth number in mammals by investigating the signals that determine whether a tooth is replaced or not. For this, we will study replacement in an animal that does not replace its teeth (the mouse) and a mammal that replaces some of its teeth (the opossum) and compare to a reptile that constantly replaces its teeth (the corn snake) and a reptile that only has one set of teeth (the chameleon). We have previously shown that in the mouse the first tooth inhibits the formation of a replacement tooth. Here we ask what those signals are (Aim 1). We know that neighbouring teeth in the opossum have different replacement capacity but we don't know what drives these differences, to make one tooth replace and one tooth not (Aim 2). Snake teeth replace each other in a tightly packed chain, so the inhibition observed in mammals doesn't occur. What changed during the evolution of mammals? Are the signals that restrict tooth number in mammals shared across toothed animals or are the rules for tooth replacement distinct in mammals compared to reptiles (Aim3). This knowledge will provide the foundation for an understanding of the mechanisms that control tooth replacement strategies, providing the possibility of controlling tooth numbers in the future. For example, if we understood how the first tooth inhibits the replacement tooth and causes its deterioration, we could prevent this from happening and reawaken tooth replacement potential. This will be particularly important in instances where non-replacing permanent teeth deteriorate prior to the end of an animal's lifespan.

In this application, we take an unbiased data-driven approach to understand the signals that control tooth replacement.
In Aim 1A, we use an established slice culture system and RNAseq to create datasets comparing murine successional dental laminas cultured in the presence or absence of the first tooth. In Aim 1B we then use our existing transgenic Wnt reporter mice to test the impact of manipulation of target pathways on the fate of the rudimentary successional dental lamina, again using our culture system.
In Aim 2, we again use RNAseq to create datasets comparing the successional dental lamina from a tooth that replaces (3rd premolar) with a tooth that doesn't replace (canine) using Monodelphis domesticus as a model. Monodelphis neonates are available through a collaboration Dr James Turner at the Crick Institute. To understand the relative impact of the first tooth at these two dental positions, we will use a culture approach to manipulate the opossum tooth germs.
In Aim 3, we will use a published dataset generated in Pogona viviceps, combined with the datasets from Aim 1 and 2, to assess similarities and differences in replacing and non-replacing teeth. Gene and protein expression analysis will then be carried out on Elaphe guttata and Chamaeleo calyptratus embryos and juveniles, followed by manipulation in culture. Elaphe guttata embryos are available from Prof Tucker's colony at Guy's. Chamaeleo calyptratus specimens are available from the collaborator Dr Buchtova.
For each aim gene and protein expression will be compared using RNAscope Multiplex Fluorescent v2 assay for co-detection of RNA and proteins, which allows the expression of several genes or protein to be compared in the same tissue.
Finally, a model of tooth replacement will be created based on the generated RNAseq data, tested by the culture experiments.