Fig. 4

Biomineralization of the notochord sheath is impaired upon chordoblast-specific cell ablation or RA reception inhibition. (A-D) Lateral views of notochords at the level of centra 3-5 in control-treated (A, n=42), RA-treated (B, n=25), Mtz-treated (C, n=63/63) and RA+Mtz-treated (D, n=47/47) R2-col2a1a:EGFP; col2a1a:CFP-NTR double transgenics at 8 dpf; confocal projections with merged green (GFP), magenta (Alizarin Red), blue (CFP-NTR) and transmitted light channels; panels A′ and C′ show single channels of middle parts of A and C, respectively. (E-G) Transmission electron micrographs of transverse sections through the notochord sheath at the level of chordacentra of untreated larvae at 4 dpf (E, n=5) or 8 dpf (G, n=5), and of Mtz-treated R2-col2a1a:EGFP; col2a1a:CFP-NTR double-transgenic at 8 dpf (F, n=6). The final width of the notochord sheath has almost been reached at 4 dpf and is not significantly affected by chordoblast ablation via Mtz administration between 4 and 6.5 dpf (compare F and G). (H-K,M) Lateral views of notochords from larvae with the indicated genotypes at 7.5 dpf, levels of centra 1-6, anterior to the left. (H-J) Projections of confocal stacks with merged magenta (Alizarin Red), green (transgene-encoded EGFP-dnRARa) and transmitted light (H, n=25; I, n=14/14; J, n=45/47) channels. Asterisk in I indicates region devoid of EGFP-dnRARa-positive cells that corresponds to centrum #5 in wild type (asterisk in H). Inset in J shows a magnified view of a portion of the notochord with gained intensity of the green channel, revealing nuclear EGFP-Hs-dnRARa fusion protein. (K,M) Stacks of merged yellow (transgene-encoded YFP) and transmitted light (K, n=38; M, n=42/42) channels. (L,N) Immunofluorescence staining on transverse sections through notochords at level of chordacentra from specimens with genotypes as in K and M and with antigen labeling as indicated. Scale bars: 0.5 µm (E-G); 50 µm (A-D,H-K,M); 20 µm (L,N). Arrows in L and N point to chordoblast layer, arrowheads to labeled cells outside the notochord [WT, n=6; Tg(col2a1a:EGFP-dnRAR), n=4; ratio of Tg(cyp26b1:YFP)-positive cells inside and outside the notochord: 26/10 (WT), 3/16 (Tg)]. AR, Alizarin Red; nc, notochord; ns, notochord sheath.