RNA-Binding Protein Hermes/RBPMS Inversely Affects Synapse Density and Axon Arbor Formation in Retinal Ganglion Cells In Vivo

Hermes is expressed in differentiating retinal ganglion cells and their axons

Both Hermes genes are specifically expressed in the RGCs of the retina in several vertebrate species (www.genepaint.org) (Gerber et al., 1999; Piri et al., 2006; Kwong et al., 2010), suggesting an evolutionarily conserved function. To gain insight into the conserved role of Hermes, we therefore characterized Hermes expression and function in two evolutionary diverged vertebrate species, Xenopus laevis and zebrafish. The genome of Xenopus laevis, like humans, contains both paralogs of the Hermes genes (rbpms and rbpms2). The coding regions of the xHermes genes are 94% identical, and are therefore considered to encode functionally redundant proteins (Zearfoss et al., 2004). Zebrafish have just one Hermes gene (rbpms2) but in two versions, hermes1a and hermes1b. Nucleotide alignment shows a 98% similarity, and the genes have identical expression patterns in the developing retina, suggesting that they also act redundantly. We hereafter refer to both proteins jointly as Hermes. To detect Hermes protein, we used an antibody raised against a conserved peptide in the Xenopus and zebrafish Hermes proteins (Zearfoss et al., 2004). This antibody detected two distinct bands on Western blots of Xenopus eye protein lysates (Fig. 1A), the lower band at ∼25 kDa corresponding to the monomeric Hermes protein with a calculated molecular weight of 22 kDa. The upper band at ∼50 kDa may indicate the presence of Hermes dimers, as reported by Gerber et al. (1999).

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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.

To gain insight into where Hermes might act in RGCs, we characterized its expression at both the RNA and protein levels during eye development in Xenopus and zebrafish (Fig. 1). ISH on Xenopus retinal sections revealed that hermes mRNA first appears in the RGC layer in the central retina at Stage 32, shortly after the onset of RGC differentiation (Fig. 1B). Further differentiation of the neural retina is accompanied by an increasingly stronger ISH signal in RGCs, extending from central to peripheral retina (Fig. 1B′,B″).

The retinotectal map first forms at Stages 37/38–40 and, before this (Stages 32–37/38), RGC axons are navigating through the optic pathway (Grant and Rubin, 1980; Holt and Harris, 1983). Hermes protein is weakly detectable in eye lysates at Stage 35/36 (Nieuwkoop and Faber, 1967), but is much stronger at Stage 40, soon after RGC axons have entered the optic tectum (Fig. 1B). Immunostaining of Xenopus retina shows that Hermes protein is first detected at Stage 35/36 (Fig. 1C,C′), in agreement with the Western blot data (Fig. 1A), indicating that protein expression begins shortly after (∼3 h) the onset of mRNA transcription at Stage 32 (Fig. 1B). The intensity of Hermes immunoreactivity increases progressively with age, with stronger expression during the later stages of RGC development (Stage 40; Fig. 1C–C″), when RGC axons have begun to arborize and form synaptic connections in the tectum.

Immunostaining of cross-sections of Stage 37/38 Xenopus embryos revealed a positive signal in the optic nerve head (ONH) and in the region of the optic tract (OT; Fig. 1D,D′), suggesting that Hermes protein is transported into RGC axons. To verify this, immunohistochemistry was performed on Stage 35/36 retinal cultures (cultured for 24 h, equivalent to Stage 41 in vivo), where single axons can be visualized. RGC axons and growth cones exhibited punctate immunostaining (Fig. 1E), reminiscent of RNP particles (Kiebler and Bassell, 2006). The signal localized to both the central and peripheral domains of growth cones and often extended into filopodia (Fig. 1E). These data indicate that Hermes, potentially along with its target mRNA cargo, is trafficked to the tips of retinal axons.

In zebrafish, hermes expression in the retina also follows the differentiation of RGCs from an early stage [32 h postfertilization (hpf); Fig. 1F,F′]. A developmental ISH series of both hermes1a and 1b performed on whole-mount eyes shows that hermes expression starts in a ventronasal patch of the retina and expands during the subsequent 24 h in a wavelike pattern across the retina, similar to the onset and differentiation pattern of RGC progenitors during the same time period (Fig. 1F–H′) (Hu and Easter, 1999; Neumann and Nuesslein-Volhard, 2000). Immunocytochemistry shows that Hermes protein expression follows the exit of RGCs from the cell cycle with a delay of only a few hours in zebrafish (Fig. 1I–K′), as in Xenopus.

Since the onset of hermes mRNA expression occurs early during RGC differentiation, we tested whether hermes expression is dependent on RGC differentiation. In the zebrafish lakritz/atoh7 mutant, RGCs fail to differentiate (Kay et al., 2001), and in the mouse, hermes expression is downregulated in atoh7 knock-outs (Mu et al., 2005). We found a complete loss of Hermes protein in lakritz fish (Fig. 1L–L″), consistent with RGC-specific expression.

Hermes RNA binding domain interferes with axonal branching

To investigate whether Hermes plays a role in RGC axon development, we first disrupted Hermes function in Xenopus by expressing a myc-tagged C-terminal truncated Hermes construct lacking amino acids 111–189, but still containing the N-terminal RNA binding RRM domain (Fig. 2A). Since the RRM domain binds to mRNA, this truncated Hermes protein (RRM) is predicted to act as a dominant negative, as it should still bind target mRNA(s) but lack other essential functions such as transport and localization. Consistent with this, the RRM-myc deletion mutant shows an altered pattern of subcellular distribution (Fig. 2B–C′). While the myc-tagged WT Hermes exhibits a punctate granular distribution in the axon and growth cone (Fig. 2B′), the myc-tagged RRM domain is not punctate, but dispersed throughout the cytosol of the axon and growth cone (Fig. 2C′).

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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.

To assess whether the expression of RRM caused an axonal phenotype in vivo, we electroporated an eGFP-tagged RRM (eGFP-RRM) construct into the eye primordium of Stage 26 embryos (Falk et al., 2007) and subsequently analyzed the optic projection at Stage 43, when the axons have reached the tectum and started to branch. The trajectory of axons along the pathway (optic tract) to the tectum was normal (Fig. 2D,D′), indicating that the long-range guidance of RGC axons to the tectum was unaffected. Nor was there any delay in axonal growth, as the length of RRM-expressing axons did not differ from controls at Stage 37/38 (control, 70.7 ± 0.9%; RRM, 70.2 ± 1.3%; p > 0.05, Mann–Whitney test; Fig. 2D″). Similar results were obtained with a C-terminal GFP-tagged RRM construct, showing that the position of the GFP-tag does not affect the result.

RRM-expressing retinal axon terminals in the tectum (Stage 43) show a severe reduction in arbor complexity (Fig. 2E–F′). Quantification revealed that the number of branches per axon was reduced from 29 (±4) in control GFP-expressing axons to 8 (±2) in eGFP-RRM axons (p < 0.0001, Mann–Whitney test; Fig. 2G). The proportional reduction in branch number is most pronounced in branches of higher order, with tertiary branches only making up 8.8% of the total amount of branches in RRM-expressing embryos, compared to 13.8% in control, and quaternary branches being reduced by >60% (Fig. 2H). The complexity of the arbors was measured by calculating the ACI (Fig. 2I) (Marshak et al., 2007). The ACI for RRM-expressing embryos confirmed a highly significant reduction in the complexity of axonal arbors compared to control embryos (p < 0.0001, Mann–Whitney test; Fig. 2J). The reduced complexity of RRM-expressing axons is particularly striking when dividing the arbors into complex (>1.4) and simple arbors (<1.4), showing that 88% of all arbors are simple or unbranched, compared to only 19% in control embryos (Fig. 2K).

Morpholino knockdown of Hermes inhibits axonal branching

As RRM domains are found in multiple RNA-binding proteins, the above-described approach may not demonstrate a specific role of Hermes in regulating branching. We therefore targeted Hermes for knockdown using translation blocking antisense morpholinos against either Xenopus or zebrafish Hermes genes. Translation MOs were designed against both hermes 1 and 2 for Xenopus (Fig. 3A) and hermes 1a and hermes 1b for zebrafish (Fig. 3B). In Xenopus, we injected 5 ng of FITC-labeled HeMO (2.5 ng of each) or CoMO at the eight-cell stage and let the embryos develop to Stage 35/36 or Stage 40 (Fig. 3C,D). Immunostaining of retinal sections in these embryos revealed an almost complete absence of Hermes signal at Stage 40 in HeMO-, but not in CoMO-injected embryos, confirming the efficacy of the HeMO (Fig. 3C′,D′). At this concentration, neither HeMO nor CoMO had any adverse effects on early axon outgrowth as assessed by acetylated tubulin staining on eye sections of Stage 40 morpholino-injected embryos (Fig. 3C″,D″). Similarly, injection of 16 ng HeMO (8.0 ng each of 1a and 1b) into one-cell zebrafish embryos reduced Hermes staining in the retina (Fig. 3E,F) without altering expression of the RGC marker zn5 (Fig. 3E′,F′) or retina morphology (Fig. 3E″,F″). To determine whether neural differentiation was affected, we investigated the expression of several retinal cell differentiation markers for aberrant expression in zebrafish embryos. For prox1 (horizontal cells), islet1 (RGC, horizontal and biopolar cells), and sox2 (amacrine cells), no significant delays or changes in expression pattern could be observed in HeMO versus CoMO-injected retinas (Fig. 3G–I′). Furthermore, staining with an antiactivated caspase-3 antibody indicated no increase in apoptosis in morphant retinas (Fig. 3J,J′). The dendrites of RGCs laminate in the inner plexiform layer (IPL). To determine whether RGC dendrite formation and lamination was disrupted in Hermes-depleted embryos, we used a transgenic zebrafish line that expressed RFP under the atoh7 promoter (Zolessi et al., 2006), thereby strongly labeling all RGCs and their dendrites. In 5 d postfertilization (dpf) embryos, we observed no gross abnormalities in outgrowth or lamination pattern of RGC dendrites in the IPL in Hermes-depleted embryos compared to control-injected embryos (Fig. 3K,K′).

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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.

To visualize single RGC axons for arborization analysis in morpholino-injected Xenopus embryos, mCherry or RFP was electroporated at Stage 26 or lipofected at Stage 19 into MO-positive retinas. Embryos were then allowed to develop until Stage 43 for analysis. Both approaches, as in RRM-injected embryos, showed that long-range axon pathfinding was unaffected in HeMO compared to CoMO-injected embryos (Fig. 4A,B). Moreover, no change in normalized brain size or axon bundle length was observed (Fig. 4C). Strikingly, we found that axonal branching was significantly compromised upon Hermes knockdown (Fig. 4D–E′). The branch number of Hermes-depleted axons was significantly reduced to 15 (±1.3) compared to 25 (±3.3) branches per axon in control embryos (p < 0.01, Mann–Whitney test; Fig. 4F). The reduction in arbor complexity is evident on all branch orders, leading to a proportional reduction of secondary, tertiary, and quaternary branches (Fig. 4G). Reduction in axonal arbor complexity is also reflected by a significant reduction in the ACI of HeMO-injected embryos (1.33 ± 0.04) compared to control embryos (1.51 ± 0.07; p < 0.05, Mann–Whitney test; Fig. 4H), and when comparing the number of complex axon arbors (>1.4) and simple arbors (<1.4) in CoMO- and HeMO-injected embryos (Fig. 4I).

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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.

Injection of HeMO in zebrafish embryos did not affect long-range guidance (Fig. 5A,A′), brain size, or axon length (Fig. 5B), as in Xenopus. To visualize individual arbors in the tectum, embryos were coinjected with an atoh7:GAPGFP reporter construct (see Materials and Methods) and allowed to develop until 4 dpf. Similar to observations in Xenopus embryos, HeMO-injected embryos showed a significant reduction in axonal branching in the tectum compared to control-injected embryos (Fig. 5C–D′). The number of axonal RGC branches was significantly reduced compared to control embryos (control, 7.35 ± 0.604; HeMO, 4.64 ± 0.477; p < 0.0001, Mann–Whitney test; Fig. 5E), and the reduction was significant in arbors of all branch orders (Fig. 5F). The decrease in axonal branching is also reflected by a reduction of the ACI in Hermes morphants (control, 1.36 ± 0.036; HeMO, 1.21 ± 0.03; p < 0.01, Mann–Whitney test; Fig. 5G), and when comparing the percentage of complex and simple arbors (Fig. 5J). Furthermore, there was a significant reduction of total arbor length (control, 102.9 ± 7.3; HeMO, 81.6 ± 6.2; p < 0.05, Mann–Whitney test; Fig. 5H) and total arbor area (control, 709.7 ± 63.7; HeMO, 501.5 ± 56.7; p < 0.001, Mann–Whitney test, Fig. 5I) in the Hermes morphants compared to control embryos, which correlates well with the observed reduction in branch number and complexity. Together with the Xenopus data, these results strongly suggest that Hermes plays a consistent and conserved role as a positive regulator of axon arborization in the optic tectum.

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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.

Branch formation is reduced in Hermes morphants

Axonal branching is a highly dynamic process, characterized in zebrafish by an early phase of branches rapidly extending and retracting, followed by a later, more stable phase with less dynamic arborization (Stuermer, 1988; Campbell et al., 2007). To investigate how Hermes depletion affects branch dynamics, we performed time-lapse imaging of GFP-labeled axons at 3 dpf (high branch dynamics) and 4 dpf (low branch dynamics) in zebrafish embryos at 15 min intervals for 30 min. As reported previously (Stuermer, 1988; Campbell et al., 2007), axons of control-injected embryos display a highly dynamic pattern, with new branches being added and retracted during the 30 min time span at 3 dpf (Fig. 6A–A″), whereas less dynamic changes are observed at 4 dpf (Fig. 6C–C″). To investigate whether the reduction in total branch number observed in the Hermes morphants is due to a change in branch dynamics, we analyzed the branches being added and retracted, respectively, during a 30 min period. At both 3 and 4 dpf, Hermes morphants added both a smaller number and a smaller percentage of branches compared to the number or branches at the start time than control embryos, although this trend was significant only at 4 dpf (Fig. 6A–E). This reduced rate of branch addition leads to reduced growth in total arbor length at both 3 dpf (control, 114.7 ± 6.3%; HeMO, 97.6 ± 4.3%, p < 0.05, Mann–Whitney test; Fig. 6G) and 4 dpf (control, 106.8 ± 3.9%; HeMO, 95.1 ± 3.7%; p < 0.01, Mann–Whitney test; Fig. 6G). Hermes-depleted embryos also show a reduction of transient filopodia that is significant at 3 dpf but not at 4 dpf (Fig. 6H). Interestingly, there was no difference in the percentage of retracted branches (Fig. 6A–D′,F) so an RGC axonal branch once formed in a Hermes morphant RGC has the same chance of surviving for 30 min as a branch from a control RGC. These results suggest that branch initiation and formation is reduced by Hermes depletion, but branch stability is not.

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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.

Synaptic density is increased in Hermes-depleted embryos

Synapse formation and arborization occur during the same developmental time frame (Alsina et al., 2001; Meyer and Smith, 2006), and many branch-regulating molecules, such as Netrin-1, BDNF, and Slit-1, also have an effect on synaptogenesis (Campbell et al., 2007; Marshak et al., 2007; Marler et al., 2008; Manitt et al., 2009), suggesting that the two processes are linked. Therefore, we next investigated whether Hermes depletion also affects synapse formation. To visualize putative synapses, we used a syn-RFP construct that labels presynaptic terminals (Meyer and Smith, 2006), together with atoh7:GAPGFP to visualize single arbors (Fig. 7A–B′). At 4 dpf, the syn-RFP signal was present as fluorescent puncta in CoMO-loaded axons at a density similar to that described previously (Fig. 7A,A′,C) (Meyer and Smith, 2006). In contrast, at 4 dpf in HeMO-loaded axons, when the reduction in branch number is most apparent, the density of syn-RFP puncta per unit length of axon terminal was significantly higher than in controls (control, 0.33 ± 0.02/μm; HeMO, 0.38 ± 0.02/μm; p < 0.05, Mann–Whitney test; Fig. 7C).

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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.

The inverse relationship between punctum density and arbor size seen above might suggest that the RGC has some kind of homeostatic control over the total number of synapses it is forming. We therefore counted that total number of syn-RFP puncta for each arbor. In line with the above hypothesis, we found that there was no significant difference between Hermes-depleted and control axons (control, 27.94 ± 2.447; HeMO, 27.09 ± 2.562; not significant, Mann–Whitney test; Fig. 7D). Thus, despite being smaller, Hermes-depleted axon arbors still contain the normal number of syn-RFP puncta.

If this inverse correlation is indicative of a homeostatic regulation of total synapse formation, it should be seen not only when comparing Hermes morphant arbors to controls, but also within control and morphant arbors of various sizes. We therefore examined the correlation between arbor size and syn-RFP density at 4 dpf in both control and Hermes-depleted embryos. We found a strong continuous inverse correlation between arbor size and syn-RFP density at 4 dpf (CoMO, r = −0.65, p < 0.0001; Fig. 8E; HeMO, r = −0.40, p < 0.02; Fig. 8F).

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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.

Hermes knockdown leads to enhanced early visual behavior

We next asked what effect the changes in synaptic density and arbor size might have on visual behavior by testing the response of control and Hermes morphant embryos in an OMR assay. Zebrafish embryos were placed in a rectangular transparent tank on a computer monitor and subjected to a 30 s duration stimulus of black/white stripe grating motions (Fig. 8A). Zebrafish larvae normally follow the direction of the moving stripes and accumulate at one end (target zone) of the tank. A positive response was scored for each fish within the target zone at the end of the stimuli, with the random chance value of occupying the target zone being 20% (Fig. 8A). WT fish start responding to this assay at 4 dpf, and by 5 dpf, >60% of WT embryos accumulate in the target area after a 30 s grating stimulus (Fig. 8B). There is no such accumulation when lakritz mutants lacking RGCs are used, or when a static rather than a moving grating is shown to WT fish (Fig. 8B). Surprisingly, when Hermes-depleted embryos were tested at 4 dpf, they showed a significantly stronger visual response compared to control-injected embryos (control, 31.06 ± 2.99%; HeMO, 49.46 ± 3.31%; p < 0.001, Student's t test; Fig. 8C). At 5 dpf, when the visual field was more fully formed, Hermes-depleted embryos responded at a similar level as control embryos (control, 52.57 ± 5.47%; HeMO, 51.84 ± 4.08%; p > 0.05, Student's t test; Fig. 8C). These data suggest that the increased synaptic density seen in Hermes morphant zebrafish at 4 dpf is functional.