Localization of transgenic node of Ranvier reporters along unmyelinated and myelinated axon stretches

(A) Schematic of node of Ranvier reporter constructs used.

(B) Confocal images of nodal reporters along individually labeled myelinated axons in transgenic animals that have all myelin labeled. Scale bar, 2 μm. (B′) Fluorescence intensity traces of each reporter around the node. Bold lines represent mean, shaded areas SD; n = 3/3 (Nfasca), 4/3 (Cntn1a), and 5/4 (NaV-II-III) animals/technical replicates.

(C) Individually partially myelinated axon at 8 dpf co-expressing cntn1b:Nfasca-EYFP in a full transgenic myelin reporter background. (C′) Magnification of the boxed area in (C). Arrowheads indicate Nfasca-EYFP clusters along unmyelinated axon parts. Arrows point to heminodal Nfasca-EYFP. Scale bars, 10 μm.

(D) Fluorescence intensity quantifications at different regions. Median ± IQR (Kruskal-Wallis test with multiple comparisons); n = 7/7 animals/technical replicates.

See also Figure S1 for supporting information.

Clusters of neuronal neurofascin are stable over time and do not co-localize with processes of oligodendrocyte precursor cells

(A) Individual unmyelinated axon expressing Nfasca-EYFP at two different time points. Arrowheads depict stable clusters and the arrow points to a cluster that disappears between time points. Scale bar, 20 μm.

(B) Quantification of Nfasca cluster and node motility. Median ± IQR (Mann-Whitney test); n = 5/5 (cluster motility) and 9/6 (node motility) animals/technical replicates.

(C) Frequency of cluster disappearance.

(D) Axon expressing Nfasca-EYFP in a transgenic animal that has all OPCs labeled. Scale bar, 50 μm. (D′) (Left) Magnification of the boxed area in (D) showing OPC processes in relation to Nfasca-EYFP accumulations over time. Scale bar, 10 μm. (Right, i–iii) Examples of possible cluster-OPC process interactions. Scale bar, 2 μm.

(E) Quantification of the frequency of cluster-OPC process interactions; n = 4/2 animals/technical replicates.

See also Figure S2 for supporting information.

Clusters of neuronal neurofascin correlate with future node position and restrict myelin sheath extension

(A) Quantification of intercluster and internodal distances along single axons. Data are expressed as mean ± SD (unpaired t test); n = 4/3 animals/technical replicates.

(B) Example images of two individual myelinated areas (dashed lines) forming a node at their left side (asterisk), and the presence (top, arrowhead) and absence (bottom, arrow) of Nfasca-EYFP heminodal accumulation at their right sheath end. Pie chart shows frequency of observations; n = 5/3 animals/technical replicates. Scale bars, 10 μm.

(C) Length of Nfasca-EYFP positive and negative sheath ends. Data derived from both in vivo imaging (left) and imaging of whole-mount immunohistochemistry (right). Median ± IQR (Mann-Whitney test); n = 8/2 (live imaging) and 7/1 (histochemistry) animals/technical replicates.

(D–D‴) Individual axon expressing Nfasca-EYFP at different time points. Dashed lines indicate myelin sheaths. Arrows depict Nfasca clusters, arrowheads point to heminodes, and asterisks depict nodes of Ranvier. Scale bars, 10 μm.

(E) Possible cluster fates and quantification of frequencies observed by time lapse imaging; n = 5/3 animals/technical replicates.

(F) Modeling of cluster fates and predicted co-localization with nodes when random positioning is assumed. The shaded curve shows the probability distribution for the simulated values (gray line indicates the mean of simulated data). The light red and dark red lines indicate the quantified cluster fates analysis shown in (E).

See also Figure S3 for supporting information.

Increased and less regular nodal spacing along individual axons in nfasca^Δ28/Δ28 mutants

(A) Targeting strategy and genetic lesion in nfasca^Δ28/Δ28 mutants.

(B) Immunohistochemistry against Nfasca on spinal cord cross-sections in adult heterozygous and homozygous nfasca^Δ28/Δ28 animals. Scale bar, 10 μm. (B′) Cross-sectional views around a node of Ranvier at different z positions in a heterozygous nfasca^Δ28/+ animal. Scale bar, 1 μm.

(C) Overviews of the spinal cord in full transgenic myelin reporter lines. Arrowheads highlight gaps between adjacent myelin sheaths. Scale bar, 20 μm.

(D and E) Close-up views showing single nodes of Ranvier marked with EYFP-cntn1a and EYFP-NaV-II-III in full transgenic myelin reporter lines. Scale bar, 5 μm.

(F) Reconstructions of two CoPA neurons and their node of Ranvier positions (arrowheads) from wild type and nfasca^Δ28/Δ28 mutant animals. Scale bar, 50 μm.

(G) Average internodal distances along single axons per axon in wild type, nfasca^Δ28/Δ28, and nfasca^Δ28/Δ28 re-expressing Nfasca-EYFP in single axons. Median with IQR (Brown-Forsythe ANOVA with multiple comparisons); n = 9/4 (wild type), 10/8 (nfasca^Δ28/Δ28), and 13/4 (nfasca^Δ28/Δ28 rescue) animals/technical replicates.

(G′–G‴) Maximum (G′), minimum (G″), and range (G‴) of internodal distances per axon as quantified in (G). Median with IQR; Brown-Forsythe ANOVA with multiple comparisons for (G′ and G‴), Kruskal-Wallis test with multiple comparisons for (G″).

See also Figure S4 for supporting information.