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The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1.
SIGNIFICANCE STATEMENT: An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting.
Figure 1. Knock-down of Hermes causes topographic guidance defects of RGC dorsal axons. Zebrafish embryos were fixed at 5 dpf and the eyes injected dorsally (D) with DiI (red) and ventrally (V) with DiO (green) to visualize the retinotectal projections (A). Whole-mount embryos injected with CoMO or HeMO were visualized in lateral (B1, B2, C1, C2) or dorsal view (B3, C3). Hermes-depleted embryos show misprojections of dorsal axons in the medial tract (C1, C2, white arrows) that are not present in embryos injected with CoMO (B1, B2). Quantifications show a significant increase of the percentage of embryos showing misprojections in the OT (D). HeMO-injected embryos show aberrant projection of dorsal axons entering the tectum through the ventral branch (C3) compared with CoMO (B3). Quantifications show that significantly more dorsal axons misroute and enter the tectum through the ventral branch in Hermes-depleted embryos compared with control (E). HeMO-injected embryos were coinjected with a construct expressing full-length myc-tagged Xenopus Hermes (He-myc; F). After 72 hpf, immunostainings show a strong myc signal in the RGC layer (RGCL). Coinjection of He-myc rescues the dorsal axons misprojections in the OT observed in HeMO-injected embryos (G), with a significant reduction of the percentage of embryos with defects (H). In HeMO-injected embryos, some dorsal axons enter in the tectum through the medial tectum (J1, J2) and this mistargeting is absent in He-myc-coinjected embryos (K1, K2). Quantifications show a rescue of misprojecting dorsal axons in He-myc-injected embryos (L). Error bars indicate SEM. Numbers of embryos analyzed are indicated on bars. Scale bars, 50 μm.
Figure 2. Hermes exerts a negative translational control of specific mRNAs in RGCs. Increase in puromycin incorporation detected by Western blotting in the Hermes-depleted condition compared with control (A, B). GFP expression is restricted to the eye in the atoh7:rpl10a-GFP transgenic line (C1, C1′), with a positive signal in Zn-5 positive RGCs and photoreceptors (C2, C3). atoh7:rpl10a-GFP embryos at 72 hpf, injected with CoMO or HeMO, were homogenized and immunoprecipitations against GFP were performed on lysates. The total RNA was then extracted from the ribosome–mRNA complexes, analyzed by bioanalyzer, and quantified by quantitative RT-PCR (D). Quantifications show an increase of nadl1.1, nadl1.2, alcama, alcamb, nrp1a, and nrp1b mRNAs bound to ribosomes in absence of Hermes (E). Quantifications of total RNAs input show no difference between CoMO and HeMO conditions (F). nrp1 in situ hybridization on 72 hpf retinal transverse sections shows no difference between HeMO- and CoMO-injected embryos (G). Quantifications of signal intensity show no difference in nrp1 expression in HeMO compared with CoMO (G). mRNA levels were calculated by using the formula 2−δδCt with β-actin mRNA as a calibrator (E, F). Error bars indicate SEM. Numbers of embryos analyzed are indicated on bars. Scale bars: C2, C3, 30 μm; G, 100 μm.
Figure 3. Hermes depletion increases Nrp1 protein in axons and induces an earlier response to Sema3A. Immunostainings on stage 32 Xenopus retinal explants show increased Nrp1 expression on Hermes-depleted growth cone compared with control (A). Quantifications show a significant increase in Nrp1 signal intensity in the growth cone at stage 28 (B), stage 32 (C), stage 35/36 (D), and stage 39 (E) embryos. Eye explants were cultured from stage 32 and 35/36 Xenopus embryos for 24 h. Sema3A was added for 10 min before fixation and the percentage of collapsed growth cones was determined (F). BSA was used as a control. Examples of collapse response of control and Hermes-depleted growth cones (G). At stage 32, Hermes-depleted growth cones display a significantly higher collapse response to Sema3A compared with control (H). This increase in collapse response is not present at stage 35/36 (I). Error bars indicate SEM. Numbers of growth cones analyzed are indicated on bars. Scale bars, 100 μm.
Figure 4. Restoring Nrp1 levels rescues the dorsal axon topography defect in Hermes-depleted embryos. Shown is Nrp1 immunostaining on retina transverse sections from atoh7:gapRFP zebrafish embryos injected with CoMO (A), HeMO (B), or HeMO+Nrp1MO (C). Quantifications show a significant increase of Nrp1 signal intensity in the HeMO condition compared with CoMO and HeMO + Nrp1MO (D). Lateral view of whole-mount DiI- and DiO-injected retina from CoMO- (E), HeMO- (F), and HeMO + Nrp1MO (G)-injected embryos. HeMO + Nrp1MO-coinjection showed a significant reduction of the percentage of embryos with mistargeting in the OT compared with HeMO (H). DiI-labelling of dorsal axons in the tectum of CoMO (I), HeMO (J), and HeMO + nrp1MO (K) injected embryos. Quantifications show a rescue of misprojecting dorsal axons in HeMO+nrp1MO injected embryos (L). Error bars indicate SEM. Numbers of embryos analyzed are indicated on bars. Scale bars: AâC, 100 μm; EâG, IâK, 50 μm).
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