Neurofascin B Is Required for Myelin Targeting in the Zebrafish CNS

(A and B) Images of wild type (A) and ue56 mutant (B) larvae at 5 days post-fertilization (dpf), showing normal morphological development of ue56 mutants. Scale bar, 500 μm.

(C–D′) Confocal images of myelin in a wildtype larva (C and C′) and ue56 mutant (D and D′) at 5dpf, visualized using Tg(mbp:EGFP-CAAX). Lateral views, with anterior to the left. (C) and (D) are maximum intensity projections of z sections taken through the entire spinal cord, with (C′) and (D′) being a projection of a subset of z sections centered closer to the midline in the region where cell bodies are prominent and myelinated in mutants. Scale bars, 20 μm.

(E) Quantitation of myelinated cell body number in wildtype and ue56 mutants at 5dpf in a 3 somite long stretch of spinal cord (wild type median = 2, 25^th percentile 0, 75^th percentile 2.5, n = 13 animals, ue56 median = 53, 25^th percentile 46.5, 75^th percentile 62.75, n = 14 animals, p < 0.0001, t test)

(F) Sequence at amino acid positions where the ue56 mutation generates a stop codon and indication of the conserved region across species.

(G) Schematic of predicted domain structure of mouse neurofascin 186, neurofascin 155, and zebrafish neurofascin B, with indication of where the mutation in ue56 mutants resides.

(H–I′) Z projections of confocal images of neurons (red) and myelin (green) in a wildtype animal (H and H′) and nfascb^ue56 mutant (I and I′), showing myelin sheaths made on axons (H and I) and myelination of cell bodies in mutants in a region with cell bodies only (H′ versus I′). Scale bars, 20 μm.

(J) Schematic cross section of the larval zebrafish spinal cord denoting areas with cell bodies (red) and myelinated axons (green) (Top panel). Dorso-ventral and medio-lateral positions of all myelinated cell bodies (dots) in a 3-somite long stretch of spinal cord of 11 mutants (bottom panel).

Ultrastructural Analyses of the nfascb^ue56 Mutant Spinal Cord

(A and B) Transmission electron microscopy (TEM) images of sections through the spinal cord of wildtype (A) and nfascb^ue56 mutants (B) at 5 dpf. Dorsal to the top, midline left. Asterisks show myelinated cell bodies. Scale bars, 5 μm.

(C–D′) TEM images of cell bodies in wildtype (C and C′) and ue56 mutants (D and D′) showing enwrapment of a cell body in the mutant with multi-lammellar myelin. Scale bars, 1 μm.

(E–H) High magnification views of myelinated axons in the dorsal (E and F) and ventral (G and H) spinal cord of wildtype (E) and mutant (F) animals. Unmyelinated axons with a diameter >0.3 μm highlighted in blue. Scale bars, 0.5 μm.

(I) Number of axons with a diameter > 0.3 μm in wildtype and nfascb^ue56 mutants (wildtype mean 114.5 ± 23.71 SD, n = 6 animals, nfascb^ue56 mutant mean 111.4 ± 23.69 SD, n = 7 animals, p = 0.8201, t test).

(J) Total number of myelinated axons in wildtype and nfascb^ue56 mutants, and numbers in the dorsal and ventral domains of the spinal cord. (Total: wildtype mean 92.17 ± 19.57 SD and nfascb^ue56 mutant mean 53.57 ± 18.20 SD, p = 0.0036, t test. Dorsal: wildtype mean 36.17 ± 7.89 SD and nfascb^ue56 mutant mean 21.93 ± 8.60 SD, p = 0.0103, t test. Ventral: wildtype mean 56.00 ± 12.52 SD and nfascb^ue56 mutant mean 31.64 ± 11.81 SD, p = 0.0041, t test. All sets wildtype n = 6 animals and nfascb^ue56 mutant n = 7 animals).

(K) Average g-ratio per animal in wildtype and nfascb^ue56 mutants at 5 dpf (wildtype mean 0.72 ± 0.01 SEM, n = 6 animals and nfascb^ue56 mutant mean 0.73 ± 0.01 SEM, n = 7 animals, p = 0.7027, t test).

(L) G-ratios of all myelinated axons assessed in the dorsal spinal cord of wildtype (black) and nfascb^ue56 mutants (red) relative to axon caliber. Each point represents a myelinated axon.

Neurofascin Functions in Oligodendrocytes to Regulate Myelin Targeting

(A) Real-time PCR analyses of the expression of nfascb, the myelin gene myrf, the neuronal genes rbfox3a (NeuN) and caspr in neuronal (N) and myelinating glial (MG) cells separated using FACS.

(B) Confocal images of Nfascb-GFP with cells counterlabelled with mRFP showing concentration of NfascB-GFP at the tips of myelin sheaths (b1-3), where paranodal junctions are localized. Scale bar, 5 μm (top panel) and 2.5 μm (b1-3).

(C and D) Confocal images of single oligodendrocytes expressing Nfascb-GFP in wild type (C) and nfascb^ue56 mutants (D). Cells expressing wild type NfascB-GFP never myelinate cell bodies, whether they are mutant or wild type. Scale bars, 10 μm.

(E–G) Analysis of myelination in mice lacking Neurofascin from oligodendrocytes.

(E and F) Confocal images of the dorsal horn where assessments for myelination of cell bodies was carried out in control (Nfasc^+/+) and Neurofascin mutants (Nfasc^−⁄−/Nfasc186), showing MBP in green, the neuronal marker NeuN in red, and DAPI to indicate nuclei in blue. Insets show single neurons, including an example of a myelinated cell body in the mutant. Scale bar, 50 μm.

(G) Quantitation of myelinated cell body number in controls and Nfasc^−⁄−/Nfasc186 mutant mice (wild type mean 0.29 ± 0.21 SD, 10 sections per mouse, n = 5 mice, Nfasc^−⁄−/Nfasc186 mean 6.51 ± 1.70 SD, 10 sections per mouse, n = 5 mice, p < 0.0001, t test).

Oligodendrocyte Neurofascin Regulates Both Myelin Targeting and Sheath Length

(A–D) Confocal images of single oligodendrocytes labeled mosaically using mbp:mCherry-CAAX in wildtype (A), two nfascb^ue56 mutants (B and C), and a heterozygous nfascb^ue56/+ animal (D). Scale bars, 10μm.

(E–G) Number of myelinated cell bodies (E) and myelin sheaths (F) and the average length of myelin sheaths (G), made by individual oligodendrocytes in wildtype, nfascb^ue56/+ hets, and nfascb^ue56 mutants.

(E) Myelination of cell bodies is increased in both nfascb^ue56/+ and nfascb^ue56 mutant oligodendrocytes (wildtype median 0.0, 25^th percentile = 0.0, 75^th percentile = 0.4, n = 28, nfascb^ue56/+ median 1.0, 25^th percentile = 0, 75^th percentile = 2.0, n = 32, nfascb^ue56 median 3.0, 25^th percentile = 1.1, 75^th percentile = 4.4, n = 24; wildtype versus nfascb^ue56/+ p = 0.0056, wildtype versus nfascb^ue56 p < 0.0001, nfascb^ue56/+ versus nfascb^ue56 p = 0.0002, all Mann–Whitney test).

(F) Number of myelin sheaths per oligodendrocyte is similar in wildtype, nfascb^ue56/+ and nfascb^ue56 (wildtype mean 12.28 ± 4.27 SD, n = 28, nfascb^ue56/+ mean 12.23 ± 3.79 SD, n = 30, nfascb^ue56 mean 12.56 ± 3.78 SD, n = 24; wildtype versus nfascb^ue56/+. ANOVA = 0.948).

(G) Average sheath length per oligodendrocyte in wildtype, nfascb^ue56/+, and nfascb^ue56 mutants (wildtype mean 30.97 ± 1.50 SEM, n = 28, nfascb^ue56/+ mean 25.75 ± 1.12 SEM, n = 30, nfascb^ue56 mean 19.46 ± 1.31 SEM, n = 24; wildtype versus nfascb^ue56/+ p = 0.0085, wildtype versus nfascb^ue56 p < 0.0001, nfascb^ue56/+ versus nfascb^ue56 p = 0.0005, all t tests).

(H) Average sheath length per oligodendrocyte in wildtype and mutant oligodendrocytes with no myelinated cell bodies (wildtype mean 32.11 ± 1.50 SEM, n = 20, nfascb^ue56 mean 21.07 ± 1.54 SEM, n = 9; p < 0.0001, Mann–Whitney test).

(I) Average sheath length per oligodendrocyte in nfascb^ue56 mutants and wildtype siblings injected with wildtype nfascb-GFP, which rescues the defect in sheath length (wildtype + nfascb-GFP mean 31.58 ± 1.41 SEM, n = 15, nfascb^ue56 + nfascb-GFP, mean 31.66 ± 1.80 SEM, n = 13, p = 0.9726, t test).

(E–I) n in all cases refers to animals.

(J and K) Confocal images of teased fiber preparations taken from wild type (K) and Neurofascin mutant (J) mice, stained with antibodies that detect Kv1.1 (red), Neurofascin 186 (green) and neurofilament 200 (red). Scale bars, 25 μm.

(L) Quantitation of myelin sheath length in Neurofascin mutant mice. Lack of Nfasc155 results in a 40% reduction in internode lengths. Wildtype mean 338.4 μm ± 6.88 SEM, n = 5 mice, Nfasc^−⁄−/Nfasc186 mean 202.1 ± 17.94 SEM, n = 5; 50 internodes per mouse, p = 0.0001, t test.

Neurofascin B Regulates Myelin Targeting during the Critical Period of Sheath Formation and Retraction

(A–A″′) Single time-point images of an individual oligodendrocyte in a wild-type animal as its myelinating processes exhibit several distinct dynamic activities over time: 1=sheath formation, 2=slow sheath growth, 3=rapid sheath growth, 4=exploratory process retraction, 5=engagement with a cell body, 6=retraction from a cell body, 7=retraction from an axon. Scale bar, 10 μm.

(B and B′) Single time-point confocal images of an individual oligodendrocyte in a wildtype animal as it is forming myelin sheaths at the beginning (A) and toward the end (A′) of the critical period. Scale bar, 10 μm.

(C and C′) Single time-point confocal images of an individual oligodendrocyte in a nfascb MO-injected animal as it is forming myelin sheaths at the beginning (A) and toward the end (A′) of the critical period. Note that a cell body has become myelinated during this time (arrows). Scale bar, 10 μm.

(D–D″′) Time series showing myelination of a cell body in a nfascb MO-injected animal. Scale bar, 10 μm.

(E–E″′) Time series showing a myelinating process being retracted from a cell body in a control. Scale bar, 10 μm.

(F) Number of myelin sheaths generated on axons per oligodendrocyte during time-lapse analyses of control and nfascb MO-injected animals (control mean 13.50 ± 2.91 SD, n = 20 oligodendrocytes from 13 animals, nfascb MO mean 12.35 ± 3.30 SD, n = 34 oligodendrocytes from 17 animals, p = 0.2041, t test).

(G) Graph showing the duration between the first formed and the last formed myelin sheath by individual oligodendrocytes in control and nfascb MO-injected animals (control mean 3.55 ± 1.45 SD, n = 13 oligodendrocytes from 10 animals, nfascb MO mean 4.11 ± 2.30 SD, n = 29 oligodendrocytes from 21 animals, p = 0.4249, t test).

(H) Time of cell body myelination during time-lapse of control (none were myelinated) and nfascb MO-injected animals. Cell bodies are myelinated by Nfasc B-depleted cells during the same period as axons.

(I) Number of myelin sheaths retracted from axons per oligodendrocyte during time-lapse of control and nfascb MO-injected animals (control mean 2.81 ± 1.60 SD, n = 16 oligodendrocytes from 13 animals, nfascb MO mean 2.81 ± 1.27 SD, n = 26 oligodendrocytes from 17 animals, p = 0.7463, Mann–Whitney test).

Neurofascin B Regulates Myelin Sheath Elongation and Stability

(A and A′) Single time-point confocal images of a myelinating process in a Tg(sox10KalTA4, UAS mEGFP) control that makes contact with a target axon (A) and transitions into a recognizable myelin sheath (A′). Scale bar, 5 μm.

(B) Average length of myelin sheaths immediately upon formation in control and nfascb MO-injected animals. Control median = 4.02, 25^th percentile 3.45, 75^th percentile 4.93, n = 91 from 13 animals and nfascb MO median = 3.67, 25^th percentile 3.10, 75^th percentile 4.63, n = 195 sheaths from 21 animals, p = 0.0611, Mann–Whitney test.

(C and C′) Confocal images of a myelin sheath elongating over time during the period of sheath formation in a control animal. Arrowheads in all panels denote ends of myelin sheaths. Scale bar, 5 μm.

(D) Average speed of individual sheath elongation in controls over a 12-h period, starting from the hour of their formation during the critical period. Each point represents a single sheath, imaged across 13 animals. No significant difference in speed of growth based on time of sheath formation, p = 0.8933, Kruskal-Wallis test.

(E and E′) Single time-point confocal images of a myelin sheath elongating over time during the critical period of sheath formation in an nfascb MO-injected animal. Scale bar, 5 μm.

(F) Average speed of sheath elongation in nfascb MO-injected animal over a 12-h period, starting from the hour of their formation during the critical period. Each point represents a single sheath, imaged across 21 animals. No significant difference in speed of growth based on time of sheath formation, p = 0.6301, Kruskal–Wallis test.

(G and G′) Single time-point confocal images of a myelin sheath shrinking over time after the critical period in a nfascb MO-injected animal. Scale bar, 5 μm.

(H) Speed of elongation of individual myelin sheaths in control and nfascb MO-injected animals for the first 6 h after their formation and the following 6 h. (control n = 34 sheaths in 8 animals, nfascb MO n = 66 sheaths in 13 animals: 0–6 h: control mean speed 0.77 ± 0.40 SD, nfascb MO mean 0.56 ± 0.41 SD, p = 0.0133, Mann–Whitney-test. 6–12 h: control mean speed 0.89 ± 0.42 SD, nfascb MO mean 0.185 ± 0.60 SD, p < 0.0001, Mann–Whitney test. Note the large number of sheaths shrinking in nfascb MO-injected animals between 6–12 h after sheath formation.

(I and I′) Confocal images of a myelin sheath elongating in a wildtype animal. Scale bar, 10 μm.

(J and J′) Confocal images of a myelin sheath elongating in an nfascb^ue56 mutant. Scale bar, 10 μm.

(K) Speed of myelin sheath elongation in Tg(mbp:EGFP-CAAX) wildtype and nfascb^ue56 mutants (control mean speed 0.34 ± 0.40 SD, n = 118 from 6 animals, nfascb^ue56 mutant mean 0.06 ± 0.23 SD, n = 215 from 9 animals, p < 0.0001, Mann–Whitney test. 16.1% in controls and 36.4% in nfascb^ue56 represent shrinking myelin sheaths.

(L) Cell body myelination in nfascb^ue56 mutants. Scale bar, 10 μm.

Caspr Regulates Myelin Sheath Elongation and Stability, but Not Targeting

(A and B) Confocal images of single oligodendrocytes labeled mosaically using mbp:mCherry-CAAX in wildtype (A) and caspr^sa12772 mutants at 5 dpf. Scale bar, 10 μm.

(C–E) Number of myelinated cell bodies (C), number of myelin sheaths (D), and the length of myelin sheaths (D), made by individual oligodendrocytes in wildtype, het, and caspr^sa12772 mutants.

(C) Myelination of cell bodies is increased slightly in caspr^sa12772 mutant oligodendrocytes (wildtype median 0.0, 25^th percentile 0.0, 75^th percentile 0.0, n = 15 animals; caspr^sa12772/+ median 0.0, 25^th percentile 0.0, 75^th percentile 0.0, n = 35, caspr^sa12772 median 0.25, 25^th percentile 0.0, 75^th percentile 1.5 , n = 25 animals; wildtype versus caspr^sa12772/+ p = 0.8056, wildtype versus caspr^sa12772 p = 0.0305, caspr^sa12772/+ versus caspr^sa12772 p = 0.0156, all Mann–Whitney test.

(D) Number of myelin sheaths per oligodendrocytes is similar in wildtype, caspr^sa12772/+ and caspr^sa12772 (wildtype mean 11.78 ± 5.14 SD, n = 15 animals, caspr^sa12772/+ mean 11.19 ± 3.26 SD, n = 33 animals, caspr^sa12772 mean 12.00 ± 3.54 SD, n = 24; ANOVA = 0.712).

(E) Myelin sheath length is reduced in caspr^sa12772 mutants (wildtype mean 29.14 ± 1.71 SEM, n = 15, caspr^sa12772/+ mean 27.58 ± 1.31 SEM, n = 33 animals, caspr^sa12772 mean 23.55 ± 1.30 SEM, n = 24 animals; wildtype versus caspr^sa12772/+ p = 0.4954, wildtype versus caspr^sa12772 p = 0.0124, caspr^sa12772/+ versus caspr^sa12772 p = 0.0374, all t test).

(F and G) Confocal images of the dorsal horn of the spinal cord in wildtype (F) and Caspr mutant (G) mice, indicating the region indicated by dashed lines wherein myelinated cell bodies were searched for. Scale bar, 50 μm.

(F′ and G′) Higher magnification views of regions within the dorsal horn of wild type (F′) and Caspr mutant (G′) animals stained with antibodies that recognize myelin basic protein (green), NeuN (red), and DAPI (blue). Scale bar, 5 μm.

(H) Graph showing that essentially no myelinated cell bodies were observed in wild type or Caspr mutant animals. Wild type mean 0.2800 ± 0.049 SEM, n = 5 (10 sections per mouse), Caspr^−⁄− 0.257 ± 0.083 SEM, n = 5 (10 sections per mouse), p = 0.8147, t test.

(I and J) Confocal images of teased fiber preparations taken from wild type (I) and Caspr mutant (J) mice, stained with antibodies that detect Kv1.1 (red), Neurofascin 186 (green), and neurofilament 200 (red). Scale bars, 25 μm.

(K) Quantitation of myelin sheath length in Caspr mutant mice. Caspr internode lengths are reduced compared to wild type; wild type mean 310.7 ± 5.32 SEM, n = 5, Caspr 226.5 ± 8.96 SEM, n = 5 animals, 50 internodes per mouse, p < 0.0001, t test.

(L and L″) Confocal images of a myelin sheath elongating in a wildtype Tg(mbp:EGFP-CAAX) animal. Scale bar, 10 μm.

(M and M′) Confocal images of a myelin sheath elongating in a caspr^sa12772 mutant. Scale bar, 10 μm.

(N) Speed of myelin sheath elongation in wildtype and caspr^sa12772 mutants: wildtype mean 0.36 ± 0.35 SD, n = 122 sheaths from 6 animals, caspr^sa12772 mean 0.16 ± 0.31 SD, n = 151 sheaths from 7 animals, p < 0.0001, Mann–Whitney test. 14.8% in controls and 28.5% in nfascb^ue56 represent shrinking myelin sheaths.