To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP-Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts. K E Y W O R D Sbone, dentin matrix protein 1, dentin sialophosphoprotein, fluorescent protein reporters, Odontoblasts

Bone sialoprotein (BSP) is a member of the SIBLING family with essential roles in skeletogenesis. In the developing teeth, although the expression and function of BSP in the formation of acellular cementum and periodontal attachment are well documented, there are uncertainties regarding the expression and function of BSP by odontoblasts and dentin. Reporter mice are valuable animal models for biological research, providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. In the present study, we examined the expression of a BSP-GFPtpz reporter mouse line during odontoblast differentiation, reparative dentinogenesis, and bone. In the developing teeth, BSP-GFPtpz was expressed at high levels in cementoblasts but not in odontoblasts or dentin. In bones, the transgene was highly expressed in osteoblasts at an early stage of differentiation. Interestingly, despite its lack of expression in odontoblasts and dental pulp during tooth development, the BSP-GFPtpz transgene was detected during in vitro mineralization of primary pulp cultures and during reparative dentinogenesis following pulp exposures. Importantly, under these experimental contexts, the expression of BSP-GFPtpz was still exclusive to DSPP-Cerulean, an odontoblast-specific reporter gene. This suggests that the combinatorial use of BSP-GFPtpz and DSPP-Cerulean can be a valuable experimental tool to distinguish osteogenic from dentinogenic cells, thereby providing an avenue to investigate mechanisms that distinctly regulate the lineage progression of progenitors into odontoblasts versus osteoblasts.

The goal of this study was to examine the effects of early and limited exposure of perivascular cells expressing α (αSMA) to fibroblast growth factor 2 (FGF2) in vivo. We performed in vivo fate mapping by inducible Cre-loxP and experimental pulp injury in molars to induce reparative dentinogenesis. Our results demonstrate that early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato cells and their accelerated differentiation into odontoblasts. In vivo lineage-tracing experiments showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp odontoblasts and devoid of BSP osteoblasts. The increased number of odontoblasts derived from αSMA-tdTomato cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.

Mild insult to dentine activated perivascular αSMA-tdTomato cells giving rise to pulp cells as well as a few odontoblasts that were integrated into the pre-existing odontoblast layer.