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Figure 5
BMP and RA signaling do not act via each other. (A–C’’) BMP-induced hyper-mineralization is not accompanied by an ectopic activation of the RA signaling reporter Tg(cyp26b1:yfp) in the notochord epithelium. Lateral views of notochords immediately dorsal to the swim bladder, anterior to the left. Images were taken by conventional fluorescent light microscopy and are shown as separate 488nm (A–C), 555nm (A’–C’) channels and with both channels merged (A’’–C’’). The cyp26b1:yfp transgene (A–C, A’’–C’’, green) labels chordoblasts and serves as a readout for RA-responsiveness. Alizarin red stains the mineralized notochord sheath (A’–C’, A’’–C’’ magenta). Note the remaining segmented YFP signal after global overexpression of BMP2b (C, arrowheads). (D–G’’) Exposure of larvae to 1 µM RA does not cause ectopic pSmad1/5/8 appearance at intervertebral positions of the chordoblast layer. Cross-sections through the abdominal notochord of 7 dpf control Tg(cyp26b1:yfp) (D–E’’), RA-treated Tg(cyp26b1:yfp) (F–F’’), and heat-shocked Tg(cyp26b1:yfp), Tg(hsp70:bmp2b) (G–G’’) larvae co-stained via immunofluorescence labeling for the activated/phosphorylated BMP signal transducer pSmad1/5/8 and for YFP, encoded by the RA responder transgene. In untreated controls, pSmad1/5/8 reactivity is restricted to vertebral segments co-expressing the RA reporter transgene (D–D’’) but absent from intervertebral spaces lacking Tg(cyp26b1:yfp) expression (E–E’’). Upon RA-treatment, such intervertebral domains (identified as such by the lack of pSmad1/5/8 activity) display ectopic Tg(cyp26b1:yfp) activity, although RA fails to induce ectopic pSmad1/5/8 reactivity (F-F’’). In contrast, as a positive control, ectopic pSmad1/5/8 reactivity in intervertebral regions, identified as such by the lack of Tg(cyp26b1:yfp) activity, is obtained by heat shock-induced ubiquitous Tg(hsp70:bmp2b) expression (G-G’’). Images represent single confocal slices shown as separate channels 555nm in magenta (D–G), 488nm in green (D’–G’) and merged with Dapi (D’’–G’’). Arrows in (D, G) point at exemplary pSmad1/5/8 positive chordoblasts. Whole figure: Genotype, age, labeling and treatments as indicated. Heat-shocks were performed once a day for 45 minutes at 40°C from 4-7 dpf. RA was applied constantly from 4-7 dpf. Scale bars: 200 µm (B); 20 µm (D’–G’). cont., vehicle-treated control; nc, notochord; HS, heat shock; RA, Retinoic acid.