Abstract
Transforming growth factor β (TGFβ) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2 cko ). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neuronal afferents. Immunofluorescence staining for β3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2 cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2 cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2 f/f DP cells. Tgfbr2 in the DP was deleted via Adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells’ ability to guide neurite outgrowth during tooth mineralization and innervation.
Highlights
The transforming growth factor β (TGFβ) superfamily regulates many developmental processes, including those that occur in the tooth and skeleton (Oka et al, 2007; Seo and Serra, 2009; Sohn et al, 2010; Wang et al, 2013; Zhen et al, 2013; Iwata et al, 2014; Choi et al, 2016; Peters et al, 2017; Niwa et al, 2018; Kawatsu et al, 2021; Zhang et al, 2021)
We subsequently assessed whether the hypothesized reduction of innervation in the Tgfbr2cko mice was due to unexpected Cre activity in neurons by crossing Osterix-Cre and ROSA26-mTmG reporter mice
We found scattered nuclear GFP in the dental pulp (DP) and membranous GFP in the odontoblast (OD) layers (Figures 1B,D), with mT isolated to DP cells without nuclear GFP (Figures 1A,D)
Summary
The transforming growth factor β (TGFβ) superfamily regulates many developmental processes, including those that occur in the tooth and skeleton (Oka et al, 2007; Seo and Serra, 2009; Sohn et al, 2010; Wang et al, 2013; Zhen et al, 2013; Iwata et al, 2014; Choi et al, 2016; Peters et al, 2017; Niwa et al, 2018; Kawatsu et al, 2021; Zhang et al, 2021). Afferent axons from the trigeminal ganglia (TG) penetrate into and throughout the tooth around the time dentin deposition begins [reviewed in (Pagella et al, 2014); (Shadad et al, 2019)]. Innervation rapidly progresses to arborize into sub-odontoblastic, odontoblastic, and predentinal axonal networks, with axons present in coronal dentin canals (Byers, 1980). This sensory innervation provides signals crucial to daily oral activities, such as eating and talking, and provides pain signals to protect the tooth organ (Hildebrand et al, 1995). Since axons from the trigeminal ganglion do not penetrate the DP until around P3 (Moe et al, 2012; Shadad et al, 2019), this lethality prevented comprehensive research into the roles of TGFβ signaling in tooth innervation
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