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Fig. 3
Chordoblasts of RA-treated zebrafish larvae acquire preosteocyte-like morphological features. (A-F) Transmission electron micrographs of transverse sections through the notochord of control- (A,C,E; level of prospective intervertebral spaces) and RA-treated (B,D,F) larvae at 8?dpf. Scale bars: 2?µm (A,B); 0.5?µm (C,D); 0.1?µm (E,F). Note the higher electron density of the notochord sheath in B and D, indicative of its strong mineralization. bv, blood vessel; cb, chordoblast; mecm, mineralized extracellular matrix; nc, notochord; ncc, notochordal cell; ns, notochordal sheath. Arrows in E point at a stretch of rough endoplasmic reticulum, which is barely found in chordoblasts of RA-treated larvae (F). (G) Quantification of notochord sheath thickness in different genetic backgrounds or after RA treatment as indicated; n=10 per condition; mean±s.d.; ***P<0.0001. dol, cyp26b1ti230g mutant (dolphin) (Laue et al., 2008). At 8?dpf (d8), the mutant shows reduced thickness of the notochord sheath, comparable to the thickness of wild types at 4?dpf (d4), most likely due to the premature reduction of sheath formation in chordoblasts. In wild-type larvae treated with RA from 4 to 8?dpf, the width of the sheath is even more strongly reduced, possibly pointing to additional RA-induced active sheath resorption (compare with Jeradi and Hammerschmidt, 2016; To et al., 2012).