Fig. S4

Chordoblast-ablated zebrafish larvae show partial and severely delayed recovery of centra formation. Lateral view of a representative untreated Tg(col2a1a:CFP-NTR) larva (A,C; n=32) and an Mtz-treated Tg(col2a1a:CFP-NTR); Tg(R2-col2a1a:EGFP) double transgenic larva (B,D; n=18) at 12 dpf (A,B) and 16 dpf (C,D; same individuals as at 12 dpf), anterior is to the left. Mineralized matrix is stained via Alizarin red (magenta). All panels represent confocal projections of merged red (here shown in magenta), green and transmitted light channels. Compared to control larvae (A) that show robust iteratively formed centra at 12 dpf none of such structures can be found in siblings of the same age in which chordoblasts have been ablated in a time window between 4 and 6.5 dpf (C). The col2a1a:EGFP transgene in the treated larvae indicates that a few chordoblasts have already recovered in some domains of the notochord (arrows) while other regions are still devoid of distinct chordoblasts (arrowheads). (B) Four days later (16 dpf), the untreated zebrafish larva has massively gained in growth and thus size of the centra, which in the meantime have formed neural arches in most rostral segments. (D) By now, also a rather regular sequence of centra has been formed in the Mtz treated specimen at those positions along the notochord where proper chordoblasts did recover earlier (white arrows). Additionally, mineralized neural arch anlagen (white arrowheads) and some associated mineralized matrix (white asterisks) can be found in adjacent caudal segments where the integrity of the chordoblast layer is still not completely given as indicated by the less intense and irregular chordoblast derived EGFP signal. Further histological analyses would have to reveal whether those mineralized matrices are of chorda- or autocentra nature. Despite this partial axial recovery, treated larvae rarely survive beyond 20dpf, possibly due to malformations of the mandibular apparatus that also expressed the col2a1a:CFP-NTR transgene during Mtz treatment. Scale bars correspond to 100μm in (A,B) and 200μm in (C,D).