Fig. 6

In mutants lacking osteoclasts, preosteocytes are normally induced, whereas osteoclast markers cannot be induced after ablation of osteogenic cells. (A,B) Fluorescent phex in situ hybridization of RA-treated pfe mutants at osteogenic front (A; of) and calvarial fragmentation site (B), counterstained with DAPI (white). pfe mutants display a similar phex expression pattern as RA-treated wild-type controls (Figs 2R vs 5L). (C) qRT-PCR analysis of osteoblast marker genes in pfe mutants and wild-type controls after DMSO or RA treatment. The preosteocyte markers phex and rankl are induced by RA in pfe^-/- mutants to a similar extent as in wild-type controls; spp1 induction is even stronger than in wild-type controls (9.5× versus 5.3×), pointing to an inhibitory role of osteoclasts on Spp1 production in preosteocytes (Sims and Martin, 2014). (D,E) In the absence of osteogenic cells, RA fails to induce osteoclastic acp5a expression in epiphyseal bar region (E), in contrast to controls containing osteogenic cells (D). (F) qRT-PCR analysis of osteoclastic marker gene expression. In the absence of osteogenic cells, RA induces a down-, rather than an upregulation of the osteoclast-specific marker gene acp5a, whereas rank levels, which are 1.56±0.13× increased upon RA treatment of wild-type controls, do not respond to RA. By contrast, ctsk expression is induced (1.2±0.02×), but induction is weaker than in controls (2.2±0.09×). The remaining ctsk and spp1 (Fig. 4K) induction is most likely due to their activation in other cell types such as macrophages, consistent with former reports (Bühling et al., 2001; Rittling, 2011). **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant.