During development, oligodendrocytes in the central nervous system extend a multitude of processes that wrap axons with myelin. The highly polarized oligodendrocytes generate myelin sheaths on many different axons, which are far removed from the cell body. Neurons use RNA binding proteins to transport, stabilize, and locally translate mRNA in distal domains of neurons. Local synthesis of synaptic proteins during neurodevelopment facilitates the rapid structural and functional changes underlying neural plasticity and avoids extensive protein transport. We hypothesize that RNA binding proteins also regulate local mRNA regulation in oligodendrocytes to promote myelin sheath growth. Fragile X mental retardation protein (FMRP), an RNA binding protein that plays essential roles in the growth and maturation of neurons, is also expressed in oligodendrocytes. To determine whether oligodendrocytes require FMRP for myelin sheath development, we examined fmr1−/− mutant zebrafish and drove FMR1 expression specifically in oligodendrocytes. We found oligodendrocytes in fmr1−/− mutants developed myelin sheaths of diminished length, a phenotype that can be autonomously rescued in oligodendrocytes with FMR1 expression. Myelin basic protein (Mbp), an essential myelin protein, was reduced in myelin tracts of fmr1−/− mutants, but loss of FMRP function did not impact the localization of mbpa transcript in myelin. Finally, expression of FMR1‐I304N, a missense allele that abrogates FMRP association with ribosomes, failed to rescue fmr1−/− mutant sheath growth and induced short myelin sheaths in oligodendrocytes of wild‐type larvae. Taken together, these data suggest that FMRP promotes sheath growth through local regulation of translation.

Myelin is a specialized membrane produced by oligodendrocytes that insulates and supports axons. Oligodendrocytes extend numerous cellular processes, as projections of the plasma membrane, and simultaneously wrap multiple layers of myelin membrane around target axons. Notably, myelin sheaths originating from the same oligodendrocyte are variable in size, suggesting local mechanisms regulate myelin sheath growth. Purified myelin contains ribosomes and hundreds of mRNAs, supporting a model that mRNA localization and local protein synthesis regulate sheath growth and maturation. However, the mechanisms by which mRNAs are selectively enriched in myelin sheaths are unclear. To investigate how mRNAs are targeted to myelin sheaths, we tested the hypothesis that transcripts are selected for myelin enrichment through consensus sequences in the 3′ untranslated region (3′ UTR). Using methods to visualize mRNA in living zebrafish larvae, we identified candidate 3′ UTRs that were sufficient to localize mRNA to sheaths and enriched near growth zones of nascent membrane. We bioinformatically identified motifs common in 3′ UTRs from 3 myelin-enriched transcripts and determined that these motifs are required and sufficient in a context-dependent manner for mRNA transport to myelin sheaths. Finally, we show that 1 motif is highly enriched in the myelin transcriptome, suggesting that this sequence is a global regulator of mRNA localization during developmental myelination.

Myelin, a specialized membrane produced by oligodendrocytes, insulates and supports axons.Oligodendrocytes extend numerous cellular processes to wrap multiple axons, and myelin sheaths differ in length and thickness. Notably, neuronal activity can modify sheath characteristics. How myelin formation is controlled at sites distant from oligodendrocyte cell bodies is not well known. Using zebrafish, we tested the possibility that 3' untranslated regions (UTRs) influence localization of myelin mRNAs, thereby enabling localized gene expression. By visualizing mRNA in living animals, we found that some candidate 3' UTRs were enriched in myelin sheaths and localized near growth zones of nascent myelin membrane. Injecting zebrafish larvae with tetrodotoxin to block action potentials reduced the amounts of mRNAs localized to myelin in a 3' UTR-dependent manner. Our data indicate that 3' UTRs contain information for neuronal activity-regulated localization of mRNAs to myelin, suggesting that changes in sheath characteristics result from mRNA regulation within nascent sheaths.Keywords: myelin, oligodendrocyte, mRNA, zebrafish 2010) and are locally translated to control axon growth and synapse formation (Kang and Schuman, 1996;Huber, Kayser and Bear, 2000;Zhang and Poo, 2002;Leung et al., 2006). Frequently, mRNA localization in neurons is determined by 3' UTRs (Taliaferro et al., 2016). RNA sequencing recently revealed that myelin purified from mouse brain has hundreds of transcripts . Many of these mRNAs encode proteins that function in cellular differentiation, translation regulation and cell-cell signaling, which might be important for sheath formation and myelination. Here we describe experiments to test the hypothesis that neuronal activity promotes myelin sheath localization of mRNAs via their 3' UTRs. RESULTS mbpa mRNA accumulates at distinct sites within nascent myelin sheathsMyelin sheaths have transcripts encoding Myelin Basic Protein (MBP) (Kristensson et al., 1986;Trapp et al., 1987;Ainger et al., 1993) but we lack information about the distribution of MBP mRNA within sheaths in vivo. We therefore began by using techniques to visualize and quantify the subcellular distribution of mRNA encoded by mbpa, a zebrafish ortholog of human and rodent MBP. First, we performed single molecule fluorescent in situ RNA hybridization (smFISH) (Femino et al., 1998;Raj et al., 2008) to detect mbpa transcripts during early stages of larval development. To mark oligodendrocytes and myelinating sheaths we used Tg(mbpa:EGFP-CAAX) larvae, which express a membrane-tethered EGFP under control of mbpa regulatory DNA. In the zebrafish hindbrain at 4 days post-fertilization (dpf), a time of active axon ensheathment and myelination (Czopka, Ffrench-Constant and Lyons, 2013), mbpa mRNA occupied cell bodies and myelin sheaths ( Figure 1A).

During development, oligodendrocytes in the central nervous system extend a multitude of processes that wrap axons with myelin. The highly polarized oligodendrocytes generate myelin sheaths on many different axons, which are far removed from the cell body. Neurons use RNA binding proteins to transport, stabilize, and locally translate mRNA in distal domains of neurons. Local synthesis of synaptic proteins during neurodevelopment facilitates the rapid structural and functional changes underlying neural plasticity and avoids extensive protein transport. We hypothesize that RNA binding proteins also regulate local mRNA regulation in oligodendrocytes to promote myelin sheath growth. Fragile X mental retardation protein (FMRP), an RNA binding protein that plays essential roles in the growth and maturation of neurons, is also expressed in oligodendrocytes. To determine whether oligodendrocytes require FMRP for myelin sheath development, we examined fmr1 -/mutant zebrafish and drove FMR1 expression specifically in oligodendrocytes. We found oligodendrocytes in fmr1 -/mutants developed myelin sheaths of diminished length, a phenotype that can be autonomously rescued in oligodendrocytes with FMR1 expression. Myelin basic protein (Mbp), an essential myelin protein, was reduced in myelin tracts of fmr1 -/mutants, but loss of FMRP function did not impact the localization of mbpa transcript in myelin. Finally, expression of FMR1-I304N, a missense allele that abrogates FMRP association with ribosomes, failed to rescue fmr1 -/mutant sheath growth and induced short myelin sheaths in oligodendrocytes of wild-type larvae. Taken together, these data suggest that FMRP promotes sheath growth through local regulation of translation.Entry clones were LR-recombined with either the pDEST-Tol2-CG2 destination vector (green heart marker; for pEXPR-myrf:FMR1-IRES-EGFP-CAAX and pEXPR-myrf:FMR1-I304N-IRES-EGFP-CAAX) or pDEST-Tol2-pA2 vector for pEXPR-sox10:FMR1-EGFP.Published plasmids: p3E-7.2sox10 [31],, p3E-EGFP [56]. Imaging and analysisWith the exception of smFISH and IHC, live larvae were imaged in all experiments. Larvae were embedded laterally in 1.2% low-melt agarose containing 0.4% tricaine for immobilization. We acquired images on a Zeiss CellObserver SD 25 spinning disk confocal system for time-lapse microscopy and cell counts (Carl Zeiss) or a Zeiss LSM 880 for all other experiments (Carl Zeiss). Images were captured with Zen software (Carl Zeiss), then processed and analyzed using Fiji/ImageJ or Zen Blue (Carl Zeiss).smFISH probe design mbpa smFISH probes were designed using the Stellaris RNA FISH Probe Designer tool by entering the zebrafish mbpa cDNA sequences obtained from Ensemble Genome Browser from transcript mbpa-206 (GRCz11). Probes with highly repetitive sequences were removed. The probes were ordered with a CAL Fluor® Red 610 Dye. Probes were resuspended in Tris-EDTA, pH 8.0 and stored at a stock concentration of 12.5 µM at -20°C.