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Fig. S1. ath5 MO does not grossly affect the lamination, position of presumptive optic nerve or neurogenesis of non-RGCs in the retina. Coronal sections through 6 dpf isl2b:GFP eyes after double-staining for GFP (B,E,H) and parvalbumin (Pv; C,F,I). (A-C) Uninjected wild type; (D-F) larva injected with control MO; (G-I) larva injected with high dose of ath5 TMO. (A′-I′) High-power views of A-I (see boxed areas in A,D,G). (A,D,G) DIC images show that retinal lamination is not affected in ath5 morphants, with the exception of a severely reduced RGC layer. (A′,D′,G′) The location of the presumptive optic nerve (on) and optic nerve head (onh, arrows), as well as a break in the RPE that represents the normal exit point for retinal axons, are clearly identifiable in ath5 morphants. (B,E,H) In controls, isl2b:GFP staining shows RGCs throughout the entire RGC layer (rgc, arrows) and their axons exiting the eye in the optic nerve (on, arrowhead). However, in ath5 morphants (H), central RGCs do not differentiate, although later-born RGCs in the periphery differentiate normally (arrow). In addition, no axons exit the eye (open arrowhead). Staining in central retina represents axons from peripheral RGCs (open arrow). GFP+ photoreceptors labeled by isl2b:gfp differentiate normally in ath5 morphants (B,E,H; small arrows). (C,F,I) Differentiation of Pv+ amacrine cells in the inner nuclear layer (a, arrows) and displaced amacrine cells in the RGC layer (da, arrowheads) appears unaffected in ath5 morphants. Scale bar: 100 µm.
Fig. S2. Eight-point scoring system used to quantify retinal axon errors. Dorsal views of retinotectal projection of 5dpf isl2b:gfp wild type and ast larvae. (A) In wild-type embryos, retinal axons always pathfind to the tectum, and errors are almost never seen. (B) In ast-null mutants, eight classes of errors are commonly seen, including midline crossing at the habenular and posterior commissures, and projections to the left and right telencephalon, diencephalon and ventral hindbrain (stars). Counting the classes of errors made by misguided axons in each larva yields an error score between 0 and 8.
Fig. S3. Background removal procedure used for transplant images. Dorsal views of projections made by donor axons in 5 dpf host larvae, control transplants. (A,B) Maximum-intensity z-projections of unedited confocal stack in wild type>wild type (A) and ast>ast (B) transplants. Autofluorescence in skin, superficial to axons, partially obscures the retinal projection. (A′,B′) Confocal projections shown after removing autofluorescence slice-by-slice by manual editing in ImageJ and Adobe Photoshop. Retinal axons are unaffected, but can be seen more clearly in the absence of skin fluorescence. Stars indicate errors (always counted in unedited confocal stacks); dotted outlines show eye positions. All transplant images in Figs 5 and 6 were processed in this manner.
Movie 1. Volume reconstruction of 72 hpf isl2b:gfp ath5 morphant eye in Fig. 2E. GFP (green) labels RGCs; lens autofluorescence has been removed by manual editing of the confocal stack. In the initial frame, dorsal is upwards, anterior rightwards and medial (i.e. the back of the eye) towards the viewer. The reconstruction is rotated first around the dorsoventral axis, then the anteroposterior axis. Axons are seen within the RGC layer, but do not exit the eye. See Fig. 2E for scale bar and other annotation.
Movie 2. Confocal imaging of a 42 hpf isl2b:gfp eye stained with ToPro3 to label nuclei and dissected away from the embryo. GFP (green) labels RGCs, whereas ToPro3 (magenta) labels all nuclei. Dorsal is upwards, anterior is rightwards. The movie starts medially and progresses laterally. The optic nerve is seen exiting the eye, and can be traced back to the RGCs. At ∼40% through the movie, a single nonretinal GFP+ neuron (presumably from the trigeminal ganglion) can be seen at the upper right. Ninety-seven serial sections spaced 1.6 µm apart; voxel size 0.5×0.5×1.6 µm. This eye had 553 RGCs (GFP+, ToPro3+ nuclei); three eyes from different 42 hpf embryos gave a mean of 625±92 (s.d.) RGCs.
Movie 3. Volume reconstruction of the 42 hpf isl2b:gfp eye, stained with ToPro3, from Movie 2. GFP (green) labels RGCs, while ToPro3 (magenta) labels all nuclei. Orientation of initial frame: dorsal upwards, anterior leftwards (reversed left-right from Movie 2). A single nonretinal GFP+ neuron (presumably from the trigeminal ganglion) can be seen.
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