Figure 4

Figure 4

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.