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Fig. S1. Validation of ephrin B3b MO knockdown. (A-C) rfng expression at 21 ss after injection of control MO (A), efnb3b-SB MO (B) or efnb3b-TB MO (C). There is a loss of boundary marker expression selectively at the r2/r3, r3/r4 and r5/r6 borders following injection of efnb3b-SB (82%, n=23) or of efnb3b-TB (82%, n=17). (D) The efnb3b gene and the different RNA products obtained following injection of control MO or efnb3b-SB MO. (E) Agarose gel electrophoresis of Ex1Fw-Ex2Re (left side) and Ex1Fw-In1Re (right side) RT-PCR products for embryos injected with Ctrl MO, efnb3b-SB (5 ng) and efnb3b-SB (10 ng). The exon 1-exon 2 RT-PCR product seen in control MO embryos is absent when injected with either concentration of efnb3b-SB (the 73 kb intron 1 is too large to amplify) and there is a concurrent increase in the amount of exon 1-intron 1 RT-PCR product.
Fig. S2. etv5b expression in rfng, sema3fb/gb and nrp2a morphants. Confocal images of dorsal views of the 30-hpf zebrafish hindbrain, anterior to the top, for (A,B) control MO, (C,D) rfng MO, (E,F) sema3fb+sema3gb MOs and (G,H) nrp2a MO. Dashed white lines indicate the position of segment borders. Left and right reconstructed lateral views (LV) plus dorsal views (DV) of etv5b whole-mount mRNA fluorescent in situ hybridisation (red), combined with HuC/D (blue) and EphA4 antibody staining (green). etv5b expression is less restricted to segment centres and/or is at lower levels in all the knockdown conditions compared with the control situation. Orientation of embryos as Fig. 1.
Fig. S3. Number of fgf20a neurons and rhombomere AP length in morphant embryos. (A) Comparison of the number of fgf20a neurons in r3-r5 in control, rfng, sema3fb+sema3gb and nrp2a knockdown embryos. There is no significant difference in the number of fgf20 neurons (average neuron number ± s.e.m.) between control embryos and the various knockdown embryos (n=7, P>0.1). (B) Comparison of rhombomere length of r3-r5 between control and rfng, sema3fb+sema3gb and nrp2a knockdown backgrounds. There is no significant difference in the AP length in r3-r5 between control embryos and the various knockdown embryos (n=4, P>0.1).
Fig. S4. Strategy to measure the distribution of fgf20a neurons following different gene knockdowns. (A) Scheme of the procedure to collect images and measure the average distance from fgf20a neuronal clusters to boundaries compared with the rhombomere centre-to-boundary distance (α), and the average fgf20a neuronal cluster length compared with the boundary-to-boundary length (β). (B-E) Examples of confocal images of fgf20a-expressing cells in 24-hpf zebrafish hindbrain, anterior to the top. All images are fitted to the same area, with its longitudinal side going from one hindbrain boundary at the bottom to another at the top (visualised by Epha4a expression, not shown). (B) Control MO; (C) rfng MO; (D) sema3fb+sema3gb MOs; (E) nrp2a MO.
Fig. S5. fgf20a and nrp2a expression colocalise. Expression pattern of (A,B) nrp2a, (C,D) fgf20a and (E,F) nrp2b. Left side of each panel shows confocal images of dorsal views of 24-hpf zebrafish hindbrain, anterior to the top. Right side shows z-reconstruction at AP positions marked by white dashed lines of the same embryo. White line delimits the neural tube position. The fluorescent in situ hybridisation signal was generated by Fast Red staining (red) and combined with Hu antibody staining (green). This approach was taken because the signals were too weak to achieve double in situ hybridisation staining. Orientation of embryos as Fig. 1.
