GoKawiilZebrafish care and use
Zebrafish were maintained in the Queen’s Medical Research Institute BVS Aquatics Facility at the University of Edinburgh with approval from the UK Home Office according to their regulations under the following project licences: 70/8436 and PP5258250, or the Vollum Institute zebrafish facility at Oregon Health and Science University (OHSU) in accordance with institutional ethical regulations and approved by the Institutional Animal Care and Use Committee at OHSU. Adult zebrafish were maintained by aquatics staff under standard conditions on a 14 h light, 10 h dark cycle. Zebrafish embryos were maintained at 28.5 °C in 10 mM HEPES buffered E3 embryo medium or in conditioned aquarium water with methylene blue. Larval zebrafish were analysed between 4–24 dpf, before sex is determined. Zebrafish larvae used for imaging experiments beyond 7 dpf were maintained at 28.5 °C in an incubator with a 14 h light, 10 h dark cycle from 1–8 dpf before being moved to tanks without water flow on a 14 h light, 10 h dark cycle until 24 dpf. In these longitudinal experiments, larvae were provided live rotifers at all times they were not embedded for imaging beyond 5 dpf (further details below).
Zebrafish stable line generation
The Tg(uas:myrEGFP-P2A-Lifeact-TagRFP) stable line was generated using Gateway cloning by combining pDestTol2PA2, p5E-10x-UAS, pME-myrEGFP and p3E-P2A-Lifeact-TagRFP. The pTol2PA2-UAS:myrEGFP-P2A-Lifeact-TagRFP plasmid (30 pg) was injected into single-cell zygotes with 12.5 pg of Tol2 transposase mRNA to generate F 0 fishes. The p3E-P2A-Lifeact-TagRFP entry vector was generated by amplifying Lifeact-TagRFP from the sox10:Lifeact-TagRFP plasmid25 with XhoI and SpeI overlap extension PCR primers and inserted into the p3E-P2A-MCS entry vector by restriction cloning.
In vivo confocal microscopy in zebrafish
To fluorescently label the myelin sheaths of single oligodendrocytes, fertilized zebrafish eggs were injected at the single-cell stage with 1 nl containing 10 pg pTol2-mbp:EGFP-CAAX, or 35 pg each of pTol2-mbp:EGFP-CAAX and pTol2-cntn1b:mCherry plasmid DNA48,64 and 25–50 ng µl−1 Tol2 transposase mRNA. In activity-dependent experiments, botulinum neurotoxin was included in the injection solution at 100 ng µl−1 as reported previously65. Zebrafish were screened to identify isolated fluorescently labelled oligodendrocytes from 3 dpf. To screen for fluorescently labelled oligodendrocytes, larval zebrafish were first anaesthetized with MS222 before mounting them in 1.5% low melting point agarose on glass coverslips. Once zebrafish were anaesthetized and mounted, oligodendrocytes in the spinal cord were selected for imaging. z-Stacks of oligodendrocytes were acquired using an LSM 880 confocal microscope with Airyscan fast mode with a 20× objective (Zeiss W Plan-Apochromat, NA = 0.8). Or using an LSM 980 with Airyscan superresolution mode with a 20× objective (Zeiss W Plan-Apochromat, NA = 1.0). z-Stacks were acquired with an optimal z-step for each experiment.
For longitudinal imaging of individual zebrafish from 3–24 dpf, Tg(sox10:KalTA4) adults were crossed to F 0 or F 1 Tg(uas:myrEGFP-P2A-Lifeact-TagRFP) adults to sparsely label oligodendrocyte lineage cells. Larvae anaesthetized with Tricaine at 3 dpf were mounted in 0.8% low melting agarose on a Petri dish and maintained in embryo medium with a low Tricaine concentration (<153 µM) and moved to an LSM 980. OPCs or newly generated oligodendrocytes (not present or existed as OPC in previous day) were imaged every 2 h until 7–8 dpf. Embryo medium was exchanged with fresh medium (lacking Tricaine) for at least 1–2 h each day before replacing Tricaine. Every other day, each larva was fully extracted from agarose and allowed to swim freely in a well of a 12-well plate with embryo medium and live rotifers for at least 2 h before being re-embedded for imaging to continue. Additional static images of the same larvae were acquired at 16 dpf and 24 dpf.
Zebrafish image analysis
For single time point analysis at 4 dpf, the number and lengths of myelin sheaths and paranodal bridges were quantified with the segmented line tracing tool in Fiji. Oligodendrocytes were analysed throughout the depth of each z-stack per cell. No cells were excluded from analyses unless there was too much myelin overlapping from neighbouring cells to reliably quantify myelin sheaths and paranodal bridges. One oligodendrocyte was analysed per zebrafish unless otherwise specified in figure legends. For long-term imaging experiments, both the green channel containing myr-eGFP signal and red channel containing Lifeact-TagRFP signal were first brightness adjusted using the Fiji Bleach Correction tool using the simple ratio function across the entire time course. Tracing of sheaths and Lifeact signal was done using the SNT plugin. Relative distance (difference in y coordinate between traces) of Lifeact signal to either the bottom or top edge of the sheath was calculated in MATLAB. The hypothetical triangular function was created with an increasing and decreasing linear array between 0.1 and 1. Cumulative sum of Lifeact movement up to 25 h post peak sheath was normalized in y from 0 to 1 with the rescale function, and in x to 101 points with imresize. For ease of viewing in plots, values for unsampled time points were filled linearly with the fillmissing function, though mixed effects models used only existing values to determine statistical significance. Linear mixed effects models used the following formula: cumulative_movement ~ time*group + (1|cell_ID) + (1|cell_ID:sheath_ID), where group consisted of anchoring segment, bridge, or bridged segment and random effects of cell ID or sheath ID were accounted for in the nesting structure. Software used for all image quantification and analysis are listed in Supplementary Table 4.
Paranodal bridge identification and quantification
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