Hörnberg H , Wollerton-van Horck F , Maurus D , Zwart M , Svoboda H , Harris WA , Holt CE .

Biotechnology and Biological Sciences Research Council , Wellcome Trust , 100329 Wellcome Trust , 085314 Wellcome Trust

	Figure 1. RGC-specific expression of Hermes in Xenopus and zebrafish. A, Western blot analysis of Xenopus embryo head lysates using an anti-Hermes antibody detects Hermes protein expression from Stage 35/36 onward. B–B′, ISH staining of Xenopus retinal transverse sections shows that hermes signal in RGCs begins at Stage 32 (B) and increases progressively at Stages 35/36 (B′) and 40 (B′). C–C′, Immunostaining detects no Hermes protein expression at Stage 32 (C) and a progressively stronger signal in RGCs at Stages 35/36 (C′) and 40 (C′) in Xenopus retinal sections. D, D′, Immunostained Hermes-positive RGC axons are visible in the ONH in the retina (C, D), in the region of the OT (D), and tectum (tec) (D′) in transverse sections through Xenopus brain at Stage 40. E, Immunostaining of Xenopus eye explants cultured at Stage 35/36 for 24 h detects strong Hermes expression in growth cones and filopodia (filo). F–H′, Whole-mount ISH staining on embryonic zebrafish eyes shows that expression of both hermes genes (Her1a and Her1b) follows the wave of RGC differentiation, beginning at 32 hpf (F, F′) and progressing across the whole retina by 39 hpf (G, G′) and 72 hpf (H, H′). I–K′, Zebrafish retinal sections coimmunostained for Hermes and zn5 (an RGC-specific marker), plus a DAPI nuclear stain, shows that Hermes protein colocalizes with zn5 staining at 32 hpf (I, I′), 39 hpf (J, J′), and 72 hpf (K, K′). L, L′, Lakritz mutant section (3.5 dpf) coimmunostained for Hermes (L) and zn5 and DAPI (L′) shows no Hermes expression in eyes or brain of embryos lacking retinal ganglion cells. Scale bars: A–A′, C, D, F–H′, 100 μm; D′, I–L′, 50 μm; E, 5 μm.
	Figure 2. Hermes RRM-domain inhibits retinal axon branching in the tectum. A, WT myc-Hermes or myc-RRM mRNA was injected into both blastomeres of two-cell stage Xenopus embryos. B–C′, In retinal growth cones (cultured from mRNA-injected embryos at Stage 37/38 for 24 h), the mutant and WT myc-tagged Hermes proteins localize differently: WT myc-Hermes (B, B′) is punctate, whereas myc-RRM (C–C′) is distributed smoothly within the cytosol. D–D″, Lateral views of Stage 37/38 whole-mount brains showing the GFP-labeled optic pathway of control- (D) and RRM-injected embryos (D′) showing no significant difference in the trajectory or the length of axon in the optic tract (D″). E–F′, Single retinal axon terminal arbors in the tectum labeled with GFP at Stage 43 in control- (E) and RRM-injected embryos (F). The corresponding outlines of each terminal arbor are shown in E′ and F′, where branches of a different order are color coded: yellow, axon shaft; cyan, primary; red, secondary; blue, tertiary. G, H, RRM-expressing terminals show a significant decrease in total branch number (G) and branches of all branch orders (H). I, J, Comparing arbor complexity using the ACI (I) revealed a significant decrease in arbor complexity and a significant increase in the number of simple arbors (J) in RRM-expressing embryos compared to control. Error bars represent SEM. **p < 0.01; ***p < 0.0001; Mann–Whitney test. The numbers of axons analyzed are indicated on the bars. Scale bars: (in B) B–C′, 5 μm; (in D) D, D′, 50 μm; (in E) E–F′, 20 μm.
	Figure 3. Hermes-depletion does not alter RGC specification and development. A, B, Translational blocking antisense MOs designed for Xenopus (A) and zebrafish (B) were used to knock down Hermes expression in the retina. C, D, Stage 40 retinal sections exhibit strong FITC-tagged MOs (green) signal throughout, indicative of robust intracellular loading following blastomere injections. C′, C″, D′, D″, MO-loaded Xenopus retinas coimmunostained for Hermes (C′, D′) and Ac-tubulin (C″, D″) show a severe loss of Hermes signal in the HeMO-loaded retina (D′) compared to control (CoMO; C′), but normal formation of the RGC layer and ONH (C″, D″). E–F″, Transverse sections of 56 hpf CoMO or HeMO injected zebrafish embryos coimmunostained for Hermes (E, F), zn5 (E′, F′), and DAPI (E″, F″). G–J′, Zebrafish retinal sections (56 hpf) of CoMO- or HeMO-injected embryos stained with antibodies to Isl1 (G, G′), prox1 (H, H′), Sox2 (I, I′), and Caspase-3 (J, J′). K, K′, Retinal sections of atoh7:RFP transgenic CoMO-injected (K) or HeMO-injected (K′) zebrafish embryos showing RGC dendrite sublamination in the IPL at 5 dpf. Scale bars: (in C, E, G) C–J′, 100 μm; (in K) K, K′, 25 μm.
	Figure 4. Hermes knockdown inhibits axonal branching in Xenopus. A–E, Embryos were blastomere injected with CoMO or HeMO and fixed at Stage 43. RGC axons labeled by eye-targeted RFP electroporation at Stage 28. Lateral view of whole-mount brains showing RFP-labeled retinotectal projections in CoMO- (A) and HeMO-injected embryos (B). There are no significant defects in long-range guidance, brain size, or axon length (C). Single RFP-labeled retinal axon terminals are highly branched in CoMO-injected embryos (D), but only sparsely branched in HeMO-injected (E) embryos. The corresponding outlines of each terminal arbor are shown in D′ and E′, where branches of a different order are color coded: yellow, axon shaft; cyan, primary; red, secondary; blue, tertiary. F–I, Hermes depletion leads to a significant reduction in average number of branches per axon (F), percentage of branches at all branch orders (G), and arbor complexity (H, I). Simple arbors, ACI < 1.4; complex arbors, ACI ≥ 1.4. Error bars represent SEM. *p < 0.05; **p < 0.01; Mann–Whitney test. The numbers of projections (C) or axons (D–I) analyzed are indicated on the bars. Scale bars: (in A) A, B, 50 μm; (in D) D–E′, 20 μm.
	Figure 5. Hermes knockdown inhibits axonal branching in zebrafish. A–B, Embryos were injected with either CoMO or HeMO and fixed at 5 dpf followed by anterograde DiI labeling of RGC axons. Control- (A) and HeMO-injected embryos (A′) at 5 dpf show no difference in long-range axon guidance, brain size, or axon length (B). C–D′, Control- (C) or HeMO-injected embryos (D) were injected with eGFP to label single axons and are shown in whole-mount views of the tectum at 4 dpf, with corresponding tracings of arbor outlines in C′ and D′. E–J, HeMO-injected embryos show a significant reduction in total number of branches per axon (E), percentage of branches at all branch orders (F), ACI index (G), total arbor length (H), arbor area (I) and percentage of complex arbors (J). Simple arbors, ACI < 1.4; complex arbors, ACI ≥ 1.4. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; Mann–Whitney test. The numbers of embryos (B) and axons (E–J) analyzed are indicated on the bars. Scale bars: (in A) A, A′, 50 μm; (in C) C–D′, 20 μm.
	Figure 6. Hermes knockdown alters branch dynamics. A–D″, GFP-labeled retinal axon arbors of HeMO- or CoMO-injected embryos were imaged every 15 min for 30 min at 3 dpf (A–B″) and 4 dpf (C–D″). E–G, HeMO-injected embryos added fewer branches at both 3 and 4 dpf, significantly at 4 dpf (E), with no difference in the percentage of deleted branches (F). Hermes morphants showed a significant decrease in total arbor length at both 3 and 4 dpf (G) and a decrease in transient filopodia at 3 dpf (H) compared to control-injected embryos. Error bars represent SEM. *p < 0.05; **p < 0.01; Mann–Whitney test. The numbers of axons analyzed is indicated on the bars. Scale bars: 10 μm.
	Figure 7. Hermes depletion causes an increase in synaptic punctum density. A–B′, Retinal axon terminals expressing GFP (green) and synaptophysin (red; syn-RFP) in the zebrafish tectum at 4 dpf. HeMO- or CoMO-injected embryos were coinjected with eGFP and syn-RFP, and the number of syn-RFP puncta was counted. C, D, HeMO-injected embryos showed a significant increase in density of synaptic puncta (puncta per square micrometer) (C), but no difference in the total amount of puncta (D). Error bars represent SEM. *p < 0.05, Mann–Whitney test. E, F, Examining the correlation between punctum density and total arbor length revealed a significant inverse correlation in both control- (E) and HeMO-injected embryos [F; r = −0.65, p < 0.0001 (CoMO) and r = −0.40, p = 0.0226 (HeMO), Pearson's correlation]. The numbers of axons analyzed are indicated on bars. Scale bar, 10 μm.
	Figure 8. Hermes morphants exhibit stronger optomotor response. A, Model of the OMR test chamber. An LCD screen underneath the transparent chamber provides the moving stimulus stripe pattern. Fish shown a nonmoving stripe pattern are randomly distributed in the chamber, whereas fish shown a directionally moving stripe pattern swim preferentially to the end zone in the direction of the motion. B, WT embryos start responding at 4 dpf, and >60% respond at 5 dpf, with Lakritz mutants and embryos showing a static pattern showing no response. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ANOVA, Bonferroni posttest. C, HeMO-injected embryos perform significantly better than CoMO-injected embryos at 4 dpf, but this difference is absent at 5 dpf. ***p < 0.001, unpaired t test. The numbers of embryos analyzed are indicated on the bars.

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