Molecular signaling during reptilian tooth development

Objectives: To study the molecular basis for tooth formation in reptiles. Reptiles from the order squamata (snakes and lizards) have teeth of similar composition to those seen in mammals. We tested the hypothesis that some of the same signaling molecules that initiate mammalian teeth also initiate first and subsequent generations of reptilian teeth.  Representative genes from each of the main classes of signals were cloned and expression analyzed with in situ hybridization. We also perform gain and loss of function experiments to determine the roles for several of these signals.

Methods: Fragments of snake (Python regius) and lizard (Pogona vitticeps)  open reading frame were amplified using degenerate PCR. Digoxygenin-labelled riboprobes were hybridized to sections. For functional experiments, jaw pieces were  placed into Trowel culture and cyclopamine was added to the media or growth factors. Following 5 days of culture, analysis of cell proliferation (PCNA antibody), apoptosis (TUNEL) and morphology was conducted.

Results: Striking localization of Shh was noted in the oral ectoderm of the python, preceding dental lamina formation. Within the tooth germs of python and pogona, there was evidence of retinoid, SHH and BMP signaling, however surprisingly, the replacement tooth rudiment was devoid of these signals. Instead expression of Lef1, a transcription factor activated by WNT signals was found in the growing tip of the second generation dental lamina.

Conclusions: Shh is required for positioning the dental lamina and initiating invagination. Both of these functions  are conserved in amniotes. Replacement tooth formation does not involve SHH, BMP or retinoid signaling however other pro-proliferative signals from the WNT family are likely to be required.

Aknowledgements: This work was funded by an NSERC grant to JMR. GH is supported by NSERC and MSFHR fellowships. JMR is a MSFHR Distinguished Scholar