Developmental suppression of schizophrenia-associated miR-137 alters sensorimotor function in zebrafish

Abstract

The neurodevelopmentally regulated microRNA miR-137 was strongly implicated as risk locus for schizophrenia in the most recent genome wide association study coordinated by the Psychiatric Genome Consortium (PGC). This molecule is highly conserved in vertebrates enabling the investigation of its function in the developing zebrafish. We utilized this model system to achieve overexpression and suppression of miR-137, both transiently and stably through transgenesis. While miR-137 overexpression was not associated with an observable specific phenotype, downregulation by antisense morpholino and/or transgenic expression of miR-sponge RNA induced significant impairment of both embryonic and larval touch-sensitivity without compromising overall anatomical development. We observed miR-137 expression and activity in sensory neurons including Rohon-Beard neurons and dorsal root ganglia, two neuronal cell types that confer touch-sensitivity in normal zebrafish, suggesting a role of these cell types in the observed phenotype. The lack of obvious anatomical or histological pathology in these cells, however, suggested that subtle axonal network defects or a change in synaptic function and neural connectivity might be responsible for the behavioral phenotype rather than a change in the cellular morphology or neuroanatomy.

Figure 1

Generation and validation of molecular tools used to transgenically inhibit or overexpress miR-137. (a) Transgene used to ubiquitously express anti-miR137 sponges (named βactin:mCherry:10 × SP137). (b) Transgene used to overexpress synthetic-miR137 (named UAS:YFPs:miR137). (c) Validation of synthetic-miR137 and anti-miR137 sponge transgenic expression/activity in 3 dpf zebrafish. Transgenic animals βactin:mCherry:10 × SP137, which ubiquitously express mCherry:10 × SP137, were injected with two plasmids simultaneously (i) 503UNC:Gal4 expressing Gal4 specifically in muscle cells and (ii) UAS:YFPs:miR137 expressing YFP fused to synthetic-miR137. Muscle-specific expression of synthetic-miR137 induced by presence of Gal4 can be tracked by the presence of YFP (processed in green in the present pictures), and correlates with downregulation of mCherry fluorescent intensity, confirming efficient activity of both anti-miR137 sponges and synthetic-miR137. Mosaic expression of YFPs:miR137 was observed as the transgenes were injected and thus not stably integrated into the genome. (d) Confocal images (0.86-μm section) showing endogenous miR-137 translational repression activity on a transcript carrying anti-miR137 sponges in 3-dpf zebrafish. The injected zebrafish (βactin:mCherry:10 × SP137; SEN:GFP) expressed mCherry:10 × SP137 ubiquitously and GFP specifically in sensory neurons (Rohon–Beard cells presented in c. These neurons also expressed endogenous miR-137 (Supplementary Figures 1 and 5). Due to the presence of miR-137, mCherry:10 × SP137 transcript translation was repressed, resulting in poor mCherry expression. Compared with MO-control, injection of MO137-02 (8 ng) dramatically increased fluorescent intensity, confirming that 8 ng MO137-02 was sufficient to inhibit endogenous miR-137 activity (quantification are presented in Supplementary Figure 5). GFP, green fluorescent protein; YFP, yellow fluorescent protein.

Figure 2

Transgenic anti-miR137 sponge expression acts in synergy with MO137-02, while transgenic pan-neuronal expression of synthetic-miR-137 rescues MO-dependent touch–response behavior. (a) Touch–response assay of wild type and transgenic βactin:mCherry:10 × SP137 zebrafish injected with MO-Control or Mo137-02. This assay allows comparing the synergistic effect of anti-miR137 sponges and Morpholinos. (b) Rescue experiments based on transgenic pan-neuronal synthetic-miR-137 expression. UAS:YFPs:miR137 fish were outcrossed with Huc:Gal4; UAS:mCherry animals and embryos were injected with MO137-02 or MO-control. Embryos were sorted at 28 hpf based on presence or absence of YFP expression, and thus on the presence or absence of synthetic miR-137. Touch–response assay was then performed using the selected animals. All experiments were performed in TAB wt background. Significantly different from control at *P<0.05, **P<0.03, ***P<0.02, ****P<0.01 (Student t-tests). MO, morpholino; YFP, yellow fluorescent protein.

Figure 3

Transient manipulation of miR-137 activity. (a) Design of morpholinos (MO) targeting the three dre-pre-miR137 copies, with mature miR-137 highlighted in yellow. Alignment was performed using CLUSTAL 2.1. (b) (i) Morphology of 72 hpf zebrafish embryos injected with MOs. (b) (ii) Morphology of 72 hpf zebrafish embryos injected with synthetic miRNAs. (c–h) Percentage of dead, malformed and normal embryos at 28 hpf following injections of MOs and synthetics miRNAs. MO137-02, MO-Control and miR-control injections were well-tolerated, while MO137-01 and miR137-mimics injections induced strong morphological defects that were most likely due to off-specific effect (see Results). These malformations included, but were not limited to, (1) heart edema, (2) curved tail and (3) abnormal trunk curvature. Percentage of normal embryos is presented in white, abnormal in light gray and dead in dark grey. (i–j) In situ hybridization against dre-miR137 at 3 dpf showing that 8 ng of MO137-02 inhibits endogenous miR-137 expression in zebrafish to a threshold that is no longer detectable even after extended revelation. miR, miRNA.

Figure 4

miR-137 knockdown impacts zebrafish touch–response behavior. (a) Touch–response assays following MO137-02 or MO-control injection. (b) Response to flash of light assay following MO137-02 or MO-control injection. (c) Rescue experiments based on miR137-mimics co-injection with MO. All experiments were performed using the Casper zebrafish strain. Different from control using t-test at *0.05, **0.03, ***0.02, ****<0.01. miR, miRNA; MO, morpholino.

Figure 5

miR-137 knockdown does not impair Rohon–Beard- (RB-), dorsal root ganglia- (DRG-), trigeminal- or Mauthner-(M-)neurons differentiation and seems to not modify their overall network. SEN:GFP transgenic animals were injected with 8–16 ng of MO-Control or MO137-02 to observe RB, M and DRG cells in vivo. No significant difference with regard to the number of cells was observed between the different conditions (Counting available in Supplementary Figure 7). No obvious difference was observed in terms of axonal network, but an in-depth analysis should be performed to conclude. Following anti-GFP immunostaining, 10 animals per conditions were analyzed at different time points using confocal microscopy and image analysis. Brightness of the original image was enhanced. GFP, green fluorescent protein; MO, morpholino.