gms | German Medical Science

Objectives: Osteocytes are key players in regulating bone mass maintenance and thus constitute a major research focus in the field of bone biology. Since analysis of osteocyte function often requires in vivo investigations, promoter elements of osteocyte-specific genes i.e. SOST or Dentin-matrix-protein 1 (Dmp1) are used to overexpress genes of interest or the Cre-recombinase for conditional deletion studies. While these tools have been very useful, emerging evidence suggests that these promoters may not be strictly osteocyte-specific, which would be critical for subsequent data interpretation. Therefore, in this study we aimed to determine the specificity of supposedly osteocyte-specific Dmp1 promoter in vivo.

Methods: To reach our goal, we crossed 8kb-Dmp1-Cre mice (i) with Ai9 tomato reporter mice (Dmp1-Cre+;Ai9T/wt), and (ii) with mice, in which the expression of the diphtheria toxin receptor (DTR) is controlled in a Cre-inducible manner (Dmp1-Cre+;iDTRT/wt) to ablate Dmp1-positive cells. Furthermore, we explored the effects of diphtheria toxin (DT) in mice harboring the human DTR (hDTR) regulated by a 10kb-Dmp1 promoter fragment (Dmp1-hDTR). Immunohistochemical staining against tomato protein was performed in tibiae and in various organs of 8-week old Dmp1-Cre+;Ai9T/wt mice and control animals. The effect of DT was determined in Dmp1-Cre+;iDTRT/wt and Dmp1-hDTR mice as well as in respective control animals by histological analyses of various organs harvested 7 days after DT treatment.

Results and Conclusion: A strong tomato expression in all osteocytes and osteoblasts covering endocortical and trabecular surfaces was observed. Furthermore, we detected tomato expression in muscle, brain and testis as well as in vessels of the heart, spleen, lung and intestine. Consistently, histological analyses of bones 7 days after DT injection into Dmp1-Cre+;iDTRT/wt and Dmp1-hDTR mice revealed partial, yet significant ablation not only of osteocytes but also of osteoblasts and lining cells. In addition, DT injection resulted in liver vacuolation, acute kidney necrosis, splenic atrophy and disturbance of bone marrow composition in Dmp1-hDTR mice, which is consistent with the expression of the hDTR in these tissues.

Our results indicate that in 8kb-Dmp1-Cre mice as well as in 10kb-Dmp1-hDTR mice, expression of the respective transgene is not restricted to osteocytes, but also occurs in other organs, some of which are known to be functionally involved in the regulation of bone homeostasis. Our findings therefore suggest that despite the great usefulness of these in vivo systems, the expression pattern of the gene of interest should be determined carefully and the results need to be interpreted accordingly.