Fig. 4

Both osteoclasts and osteogenic cells are required to mediate RA-induced calvarial fragmentation. (A,B) AR staining of SL9-10 wild-type sibling (A; n=7/7) and pfe mutant (B; n=9/9) after 7days of RA treatment. Arrows point to calvarial fragmentations in the wild type and corresponding regions in pfe mutant, which lacks fragmentation. (C-F) TRAP labeling of transverse sections through epiphyseal bar (eb) region, where RA-calvarial fragmentation occurs (see Fig. S4B). In comparison with wild-type siblings (D; n=3/3), RA treatment fails to induce TRAP activity in pfe mutant (F; n=4/4), with staining comparable to the untreated WT sibling (C; n=3/3) or mutant (E; n=3/3). (G-J) Calcein staining of osx:NTRmcherry transgenic fish after MTZ treatment (I,J) for osteogenic cell ablation, and/or RA (H,J) or DMSO (G,I) treatment. After ablation of osteogenic cells (J; n=7/7), RA fails to induce calvarial fragmentation seen in the RA only-treated transgenic fish (H; n=5/5). Also, pure ablation of osteogenic cells fails to cause calvarial fragmentation (I; n=6/6). Arrows point to calvarial fragmentations in the RA-treated, non-ablated transgenic, and corresponding regions of the other specimens lacking fragmentation. (K) Comparative qRT-PCR analysis of isolated calvarial plates from SL11-12 sibling fish containing (MTZ) or lacking (+MTZ) osteogenic cells, and treated (RA) or not treated (DMSO) with RA. Columns with same superscript letter (a,b,c) are not significantly different (P>0.05). In the absence of osteogenic cells (MTZ+RA), RA fails to significantly induce expression of phex and rankl (MTZ+RA versus MTZ+DMSO), whereas spp1 is much more weakly induced (1.8× in MTZ+RA versus MTZ+DMSO, compared with 6.6× in RA versus DMSO). The remaining spp1 induction most likely reflects expression in other cell types (compare with Fig. 6 F and see Discussion). Furthermore, RA-treated fish containing osteogenic cells (RA) display 5.5-fold higher expression levels of the direct osteoclastogenesis effector rankl compared with RA-treated fish lacking osteogenic cells (MTZ+RA). mc, melanocytes.