Transgenic Mice Expressing a Membrane-Bound GFP in Odontoblasts and Osteocytes

Recently it has been shown that osteocytes display dynamic motions by the extension and retraction of dendrites and shedding of microvesicles. These dynamic properties may be important for bone function. To further investigate the dynamic properties of these cells, transgenic mice were developed which express a membrane-targeted green fluorescent protein(GFP) driven by the dentin-matrix-protein-1 promoter (Dmp1-mem-GFP-mouse). Objectives: goals of this study were to determine the GFP expression pattern in the craniofacial skeleton/dentition of Dmp1-mem-GFP mice and characterize an immortalized osteocyte-like cell-line derived from this mouse. Methods: Mandibles from 7 day and 8 week-old Dmp1-mem-GFP mice and littermate controls were fixed and decalcified. 50um cryosections were counterstained with phalloidin (to visualize F-actin) and DAPI (to visualize nuclei). Undecalcified whole-mount-calvaria from the 7day-old mice were counterstained with DAPI and alizarin-red (to stain mineral). The samples were imaged by 3D confocal microscopy. Calvarial cells isolated from Dmp1-mem-GFP mice were immortalized by T-antigen transfection and a clonal cell-line was established.  Mineral deposition was quantified and expression of GFP, the osteoblast marker alkaline-phosphatase and osteocyte markers E11 and sclerostin were measured in this cell-line over a timecourse of 28 days. Results: Dmp1-mem-GFP was selectively expressed in calvarial and alveolar osteocytes, odontoblasts and some pulpal cells adjacent to odontoblasts. The membrane-targeted-GFP highlighted odontoblast processes and revealed abundant membrane-bound-vesicles concentrated at the dentin-enamel-junction. Characterization of the cell-line showed initial expression of the late osteoblast and early osteocyte markers alkaline-phosphatase and E11. By day 14 the cells expressed Dmp1-GFP and began to mineralize. Finally, by 28 days they expressed the late osteocyte marker, sclerostin. Conclusions: The Dmp1-mem-GFP mouse is a novel model for studying and imaging membrane dynamics/vesicle function in osteocytes/odontoblasts. The Dmp1-mem-GFP osteocyte-like cell-line recapitulates the early-to-mature osteocyte transition and provides a useful tool to study osteocyte membrane/dendrite and vesicle dynamics. Supported by UMKC Summer Scholars Program.