Analysis of DMP1-GFP transgene during odontoblast differentiation

DMP1 is a matrix protein, initially isolated from dentin that is highly expressed in osteocytes and odontoblasts. The mouse DMP-1 cisregulatory sequences have been used to drive Green Fluorescent Protein (GFP) expression in osteocytes and odontoblasts. Objective: In the present study, we have examined the patterns of expression of DMP1-GFP during odontoblast differentiation in vivo and in vitro using primary dental pulp cultures. Materials and methods: The temporal and spatial pattern of expression of GFP in the mandibular molars of DMP1-GFP transgenic mice was examined in tissue sections under fluorescence light. Primary cultures derived from the coronal portion of dental pulp from DMP1-GFP were used to: 1) examine the stage-specific activation of DMP1-GFP transgene during in vitro mineralization, 2) examine the changes in the percentage of DMP1-GFP expressing cells (GFP+) at various time points by FACScan Calibur analysis and 3) identify and isolate populations of cells from primary pulp cultures by FACS sorting based on GFP expression and examine their differentiation in vitro. Results: DMP1-GFP was expressed in the entire layer of odontoblasts covering the dental pulp. Primary pulp cultures were composed of a heterogeneous group of cells with respect to cell morphology and expression of DMP1-GFP. A small population expressed low level of DMP1-GFP prior to mineralization. The number of DMP1-GFP expressing cells increased with time in cultures and appearance of late markers of odontoblast differentiation. There was also a close correlation between the areas of the cultures expressing DMP1-GFP and areas of mineralization examined by standard von Kossa reagent and Xylenol Orange staining. Cultures established from DMP1-GFP(-) cells formed mineralized nodules and contained DSPP expressing cells. Conclusions: DMP1-GFP mice provide new experimental model to study odontoprogenitor population in dental pulp and the molecular mechanisms that regulate their progression into odontoblasts.  Supported by Grant R01-DE016689.