The RNA Binding Protein FMRP Promotes Myelin Sheath Growth

Abstract

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.