Figure 3

BMP directly targets chordoblasts to regulate Entp5a-mediated mineralization of the notochord sheath. (A–D) BMP induced hyper-mineralization of the notochord sheath requires an intact chordoblast layer. (E–N) Chordoblasts receive BMP signals as indicated by the presence of the responsible intracellular mediator pSmad1/5/8. Apart from chordoblasts, global overexpression of bmp2b via induction of Tg(hsp70:bmp2b) also resulted in the gain of pSmad1/5/8-positive cells within the myosepta, muscle tissue and the neural tube (K-N). (O–R) Mosaic chordoblast-specific blockage or activation of the intracellular BMP signaling cascade alters entpd5a expression autonomously in targeted notochord epithelium cells. (A–R) Maximum intensity projections of confocal images with merged channels showing notochords and their immediate surroundings in live whole-mounts (A–E, J, O–R) and on stained cryosections (F, H, K, M). (G, I, L, N) show corresponding single 555nm channels in magenta. (E, J) delineate overviews of those areas of heat-shocked larvae from which the sections presented in the corresponding rows were prepared. (A–P) have been assayed in the abdominal region at the level of the swim bladder, (Q) and (R) in the tail. (A–E, H–J, M–R) are lateral views with anterior to the left, (F, G, K, L) show transverse sections with dorsal upwards. Arrowheads in (F, G, I, K, L) point to exemplary chordoblasts with clear nuclear pSmad1/5/8 staining. Inset in (L) depicts magnified single channel images of pSmad1/5/8-positive chordoblast indicated in (L) by connected arrowhead. Arrows in (P) mark magenta-colored dn-bmp1b-p2a-dTomatoCAAX expressing cells that are located in a usually entpd5a-positive chordoblast cluster (compare with (O)), but that do not express the entpd5a:egfp transgene when BMP signal reception is blocked (N=5; n=28/39 dTomatoCAAX-positive, but EGFP-negative cells). Arrows in (R) point at a prominent group of chordoblasts that are triple positive for EGFP, the constitutively active BMP receptor Bmpr1b and dTomatoCAAX (N=2, 48 dTomatoCaax and EGFP double-positive of 53 dTomatoCAAX expressing chordoblasts). Ages, treatments, genotypes and color-coded markers as indicated. Scale bars: 50 µm (A, E, I, O, P); 25 µm (G). Mtz, Metronidazole; nc, notochord.