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Fig. S1. Increased migration of the primorduim after fusion with SPCs. (A) Detection of SPCs from the 10s stage. Whole-mount hmx3 in situ hybridisation at 10s and 16s in wild-type and ody-/- embryos. Anterior to the left. Black arrowheads indicate SPCs. White arrowheads show hmx3-expressing spinal cord neurons. mp, main primordium placode. Scale bar: 25 µm. (B) Cell tracking of the primordium before and after the fusion. Average speed of leading, mid and trailing primordium cells during 1 hour period before and after the fusion between SPCs and the main primordium. P values: 1=0.018, 2=0.028, 3=0.015, 4=0.0001, 5=0.040 and 6=0.258.
Fig. S2. Failure of fusion in Cxcr7b morphants reveals the dynamic behaviour of SPCs. (A) hmx3 expression at 28 hpf, showing the split phenotype observed in 10% of Cxcr7b Mo-injected embryos. (B) Images from a time-lapse sequence performed from 26s on a cldnb:lyngfp embryo injected with Cxcr7b Mo, showing changes in the shape of the SPC cluster and their protrusive activity in all directions. The migration of the main primordium is delayed but it eventually catches up the SPC cluster (supplementary material Movie 3). (C) Images from a time-lapse performed from 20s onward on another Cxcr7b morphant (supplementary material Movie 4). In both movies, no net forward or backward migration of the SPCs is observed. Scale bars: 25 µm.
Fig. S3. Fgf signalling attracts SPCs towards the primordium. Side view and anterior to the left. (A) hmx3 expression in heat-shocked hsp70:dn-fgfr1 embryos and control siblings. The position of SPCs, indicated by black arrowheads, is shifted caudally in hsp70:dn-fgfr1 embryos. Black lines indicate somitic borders. White arrowheads indicate hmx3-expressing spinal cord neurons. (B) SPC frequency at 24s in DMSO- and SU5402-treated embryos. *P=0.018. (C) Average distances covered by the tip of the main primordium (red) and SPCs (blue) in SU5402-treated embryos (n=49) and DMSO controls (n=34) at 24s. *P=0.041, ns, non significant (P=0.85). (D) Additional examples of hmx3 expression (purple) in embryos transplanted with Fgf10- or tdTomato-expressing cells (pink). Black, pink and white arrowheads show SPCs, transplanted cells, and hmx3-expressing spinal cord neurons, respectively. Dashed lines represent the horizontal somitic midlines. Focal planes focused on the primordium and SPCs (a,b) or on the transplanted cells in the neural tube (c,d). mp, main primodium; S, somite. Scale bars: 25 µm.
Fig. S4. Additional examples of the fate-mapping of SPC. (A,B) Primordium region of 18s embryo injected with Kaede mRNA, before (A) and just after (B) photoactivation of Kaede in a small circular region including putative SPCs. The main primordium is outlined with white dashed lines. (C,D) Low (C) and high (D) magnification of the same embryo at the prim-15 stage, showing red cells (white arrowhead) in the leading zone of the migrating primordium. No deposited red cells are observed between the region of activation (red arrowhead) and the primordium. (E,F) Low (E) and high (F) magnification of the same embryo at 48h, showing the presence of red cells (white arrowheads) in the last deposited neuromast and in the primordium that has reached the tail region. No deposited red cells are detected more anteriorly. (G,H) Primordium region of 18s embryo injected with Kaede mRNA, before (G) and just after (H) photoactivation of Kaede in a region containing all potential SPCs, including half of somite 1 to somite 5. The main body of the primordium is outlined with white dashed lines. (I,J) Low (I) and high (J) magnification of the same embryo at the prim-11 stage, showing red cells (white arrowhead) scattered in the leading zone of the migrating primordium. No deposited red cell can be observed between the region of activation and the primordium. (K,L) Low (K) and high (L) magnification view of the same embryo at 48h, showing a large contribution of red cells (white arrowheads) to the three posterior-most neuromasts. No deposited red cells are detected more anteriorly. nt, neural tube; op, otic placode; S, somite. Scale bars: 25 µm.
Movie 1. Time-lapse movie showing the fusion between the main body of the primordium and two SPC clusters in a cldnb:lyngfp embryo injected with mRNA encoding H2B-RFP. The primordium region is imaged at 2-minute intervals beginning at the 18s stage. Coloured dots indicate two proximal SPCs before the fusion with the main primordium.
Movie 2. Time-lapse movie on a cldnb:lyngfp embryo showing that the fusion between the main primordium and SPCs is a consequence of the forward movement of the primordium. The lower panel shows trackings of SPCs and main primordium tip cells. The primordium region is imaged at 5-minute intervals beginning at the 20s stage.
Movie 3. Time-lapse sequence performed on a cldnb:lyngfp embryo injected with Cxcr7b Mo. The migration of the main primordium is delayed but it eventually catches up with the SPC cluster. Note the highly protrusive activity of the SPCs in all directions and the absence of net forward or backward movement of the cluster before the fusion. The primordium region was imaged at 2-minute intervals beginning at the 26s stage.
Movie 4. Time-lapse sequence showing the absence of net backward or forward movement in a Cxcr7b morphant embryo. The primordium region was imaged at 5-minute intervals beginning at the 20s stage.
Movie 5. Time-lapse movie showing the backward (rostral) migration of SPCs in a ody−/− cldnb:lyngfp embryo. The lower panel shows trackings of SPCs and main primordium tip cells. Note the protrusions of the SPCs directed towards the primordium during the backward movement. The primordium region is imaged at 15-minute intervals beginning at the 20s stage.
Movie 6. Time-lapse movie showing the forward (caudal) migration of SPCs in a cldnb:lyngfp embryo treated with SU5402. The lower panel shows trackings of SPCs and tip cells of the main primordium. Note the protrusions of the SPCs in the direction of the movement. The primordium region was imaged at 15-minute intervals starting at 18s and SU5402 was applied 2 hours prior to imaging. Towards the end of the movie, the main primordium (out of focus) fuses with SPCs.
