Fig. 7

Fig. 7

Osteoclasts are tightly associated with preosteocytes and, in contrast to osteogenic cells, lack cyp26b1 expression. All images show wild-type specimens at 7dpt with RA. DMSO-treated controls stained in parallel lacked detectable spp1, acp5a and cyp26b1 signals (not shown). A-F,I-L show sagittal sections through sites of calvarial fragmentation; G,H show transverse sections at the osteogenic front region of a frontal plate, labeled by in situ hybridization with the indicated probes and/or anti-RFP immunolabeling of osteogenic cells expressing tg(osx:NTRmCherry). Osteoclasts are labeled with asterisks, preosteocytes with filled arrowheads, and other osteogenic cells with open arrowheads. (A-C) spp1 is expressed in an osx⁺ cell (preosteocyte), which is tightly associated with an spp1⁺, osx cell (osteoclast) (n=8/8). Note that particularly on the outer surface of the calvarial plate, not all osx⁺ cells express spp1. (D-F) ssp1 is expressed in an acp5a⁺ osteoclast, which is tightly associated with an ssp1⁺, acp5a cell (preosteocyte) (n=13/13). (G,H) At the osteogenic front, almost all osx⁺ osteogenic cells lining the growing calvarial plate display RA-induced cyp26b1 expression (n=5/5). (I,J) Sites of calvarial fragmentation contain both cyp26b1⁺ and cyp26b1 osteogenic cells (osx⁺). Note that chondrocytes in the epiphyseal bar lack RA-induced cyp26b1 expression (n=5/5). (K,L) acp5a⁺ osteoclasts lack cyp26b1 expression (n=8/8). Panels G-J show additional cyp26b1 expression in dermal fibroblasts, known targets of RA signaling (Nelson and Balian, 1984; Varani et al., 1990). Panels I-L show additional and very strong RA-induced cyp26b1 expression in a distinct tissue adjacent to the epiphyseal bar. The exact nature of this tissue is unknown. It contains multiple blood vessels, not seen in such sizes and densities in other subcalvarial regions (compare with Fig. S5A). bv, blood vessel; cp, calvarial plate; df, dermal fibrobasts; eb, epiphyseal bar; of, osteogenic front.