Tgfbr2 in dental pulp cells guides sensory innervation in developing teeth

Abstract

Transforming growth factor {beta} (TGF{beta}) plays an important role in tooth morphogenesis and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in the dental pulp (DP) mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2cko). These mice survived postnatally, but had significant defects in bones and teeth, including reduced mineralization and short roots. H&E staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity was localized within the tooth and was active in the DP and derivatives, but not in neurons. Previous research has established that paracrine signals from the dental pulp attract trigeminal axons toward and into the tooth during postnatal development, yet very little is known about the signals that regulate tooth innervation. Immunofluorescent staining for a neuronal marker, {beta}3 tubulin, was performed on serial cryosections from control and mutant molars at two different postnatal stages. Confocal imaging and pixel quantification of {beta}3 tubulin demonstrated reduced innervation in P7 and P24 Tgfbr2cko first molars compared to controls, indicating that the signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. Quantitative real time PCR showed that the mRNA expression levels of several neuronal genes were reduced. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2f/f DP cells. Tgfbr2 in the DP was deleted with an adenovirus expressing Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured in the presence of Tgfbr2-positive DP cells compared to neurons cultured alone. Further, axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. These results indicate that Tgfbr2 in the DP mesenchyme regulates paracrine signals that guide sensory innervation of the tooth. Note: A list of abbreviations is included in the appendix.