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1 Supplementary Figure 1 Global TeNT expression effectively impairs synaptic transmission. Injection of 100 pg tent mrna leads to a reduction of vesicle mediated synaptic transmission in the spinal cord at 4 days post fertilisation (dpf). (A) Current-clamp and voltage-clamp recordings from spinal cord neurons showing synaptic potentials and currents (excitatory inward and inhibitory outward currents) induced by stimulation (arrow) of descending axons from the brain in control animals. (B) In TeNT expressing animals no synaptic potentials or currents were induced in spinal cord neurons by stimulation (arrow) of descending inputs from the brain. (C) Quantification of the average area of the inward current induced in spinal cord neurons (clamped at 65 mv) in control animals (n = 5, one neuron per animal) and TeNT expressing (n = 5, one neuron per animal, Student s two-tailed t- test, p = ). (D) Quantification of the average area of the outward current elicited in spinal neurons (clamped at 0 mv) in control animals (n = 5, one neuron per animal) and in TeNT expressing animals (n = 5, one neuron per animal, Student s two-tailed t- test, Student s two-tailed t- test, p = ).
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3 Supplementary Figure 2 TeNT does not delay the onset of myelination or generally impair neuronal development. (A) Myelination occurs according to an anterior-posterior gradient in the developing spinal cord. Therefore the time of appearance of myelin sheaths at specific points along the A P axis serves as a proxy for the timing of developmental onset of myelination. The most posterior myelin sheath in the spinal cord at 80 hpf, myelinating the Mauthner axon, can be observed between somite in both control (top) and TeNT expressing animals (bottom) (Scale bar 45 µm). (B) Quantification of the most posterior myelin sheaths at 70 hpf (somite 5.6 +/- 1.9 in control animals n = 7, somite 4.8 +/- 2.2 in TeNT expressing animals n = 4), 75 hpf (somite /- 2.0 in control animals n = 8, somite /- 3.0 TeNT expressing animals n = 8), 80 hpf (somite /- 1.5 in control animals n = 9, somite /- 2.3 in TeNT expressing animals n = 9) (Two-way ANOVA, p = 0.68). (C) Transgenic spinal cord neurons labelled with Tg(cntn1b(5kb):mCherry) in controls (top) and TeNT expressing animals (bottom) (Scale bar 40 µm).
4 Supplementary Figure 3 Synaptic vesicle release is required for myelination of the correct axon number in the CNS. (A) Transmission electron micrograph overviews of hemi-spinal cords in control (left) and TeNT expressing (right) animals. Box indicates magnified area in the dorsal spinal cord. Unmyelinated axons > 0.3 µm diameter are coloured in turquoise. Scale bar in overviews (left) 5 µm and in insets (right) 1 µm. (B) Percentage of myelinated and unmyelinated axons > 0.3 µm per entire hemi-spinal cord (control animals n = 5, TeNT expressing animals n = 5). (C) Percentage of axons that are myelinated between µm and above 0.9µm (control animals n = 5, TeNT expressing animals n = 5). Notice that these data indicate a stronger % reduction of myelination of the smaller calibre axons, which could suggest a greater requirement for synaptic vesicle release in the myelination of small caliber axons. Alternatively myelination of axons of all permissive sizes may have the same requirement for synaptic vesicle release, but larger axons may simply have a higher probability of initial contact with oligodendrocyte processes and subsequent myelination due to their greater surface area.
5 Supplementary Figure 4 Impairment of synaptic vesicle release causes a small reduction in myelinating oligodendrocyte number. (A) Lateral view of Tg(mbp:EGFP) expressing animals (somite level 10 11), labelling the cytoplasm of differentiated oligodendrocytes in the spinal cord of control (top) and TeNT expressing (bottom) animals at 4dpf. (Scale bar 15 µm). (B) Quantification of Tg(mbp:EGFP) expressing cells in a 425 µm stretch of spinal cord from somite 8 to 11 (Student s two-tailed t-test, p = 0.044, control animals n = 40, TeNT expressing animals n = 36).
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7 Supplementary Figure 5 Impairment of synaptic vesicle release causes a small reduction in OPC number. (A) Cells of the oligodendrocyte lineage express olig2:egfp and sox10:mrfp, revealing Oligodendrocyte Precursor Cells (OPCs) at 60 hpf and both OPCs and oligodendrocytes from 72 hpf in control (top) and TeNT (bottom) animals. Arrowheads point to cells expressing both transgenic reporters. (B) OPC number at 60 hpf in a 425 µm stretch of spinal cord between somite levels 8 and 11 (82.2 +/- 12.4, control animals n = 42 vs /- 12.8, TeNT expressing animals n = 63). Student s two-tailed t-tests, p = E-05). (C) OPC and OL number at 72 hpf in a 425 µm stretch of spinal cord between somite levels 8 and 11 (98 +/- 10.5, control animals n = 33 vs /- 13, TeNT expressing animals n = 27). Student s two-tailed t-tests, p = ). (D) Number of proliferation events in a 425µm stretch of spinal cord between 60 and 72 hpf. (4.0+/- 3.2, control animals n = 6 vs 3.7 +/- 2.3, TeNT expressing animals n = 6. Student s two-tailed t-tests, p = 0.84).
8 Supplementary Figure 6 Global reduction of synaptic vesicle release does not affect average myelin sheath length. (A) Individual oligodendrocytes labelled by mbp:mcherry-caax in control (top), and TeNT (bottom) expressing animals at 4 dpf. Scale bar 10 µm. (B) Average myelin sheath length per oligodendrocyte in control and TeNT expressing animals. (Control oligodendrocytes 30 +/- 8 m vs TeNT oligodendrocytes 28 +/- 8 m. Student s two-tailed t-test, p = 0.2, control, n = 45 cells in 24 animals; TeNT, n = 47 cells in 26 animals).
9 Supplementary Figure 7 TeNT does not function in oligodendrocytes to regulate myelin sheath number. (A) Oligodendrocytes from donor embryos labelled with mbp:egfp-caax (top and middle) and sox10:mrfp (bottom) in genetic chimeras. Top shows a single oligodendrocyte in a control to control chimera, middle shows two oligodendrocytes in a TeNT to control chimera, and bottom a single oligodendrocyte in a control to TeNT chimera. (B) Myelin sheath number per cell (One-way ANOVA, p = 0.003, followed by Student s two-tailed t-tests: control to control vs TeNT to control, p = 0.5, control to control vs control to TeNT, p = 0.009, TeNT to control vs control to TeNT, p = Control to control, n = 15 cells in 4 animals; TeNT to control, n = 18 cells in 9 animals; control to TeNT, n = 13 cells in 7 animals).
10 Supplementary Figure 8 TeNT functions in neurons to regulate myelination in vivo. (A) Projections of confocal stacks showing single control reticulospinal axons labelled with TdTomato (top three rows) and TeNT toxin expressing axons (bottom three rows) labelled with a TeNT-TdTomato fusion protein in Tg(mbp:EGFP-CAAX) reporter line. Boxes in left panels indicate individual myelin sheaths shown in insets. Arrowheads point to individual myelin sheaths along the length of single axons. Arrows in insets point to the extremities of a sample myelin sheath visible in single imaging planes in the areas indicated by boxes.
11 (B). Myelin sheath number along individual control and TeNT expressing reticulospinal axons. (19.5 +/- 5.5 per mm in control neurons vs /- 5.1 per mm in TeNT expressing neurons. Student s two-tailed t-tests, p = 0.016, control n = 13 neurons in 12 animals and TeNT n = 12 neurons in 12 animals).
12 Supplementary Figure 9 PTZ augments escape response, increases sheath number per cell, and elevates oligodendrocyte number.
13 (A + B) Plots of distance travelled by representative control (A) and PTZ treated (B) zebrafish in response to touch stimuli. (C) Total distance moved per animal in response to touch stimulus (average of three touches) in control and PTZ treated animals: 64 +/- 45 mm control (n = 26) vs 95 +/- 41 mm PTZ treated (n = 30). Student s two-tailed t-tests, p = (D) Individual oligodendrocytes labelled by mbp:mcherry-caax in control (top) and PTZ treated (bottom) animals at 4 dpf. Scale bar 10 µm. (E) Myelin sheath number per cell in a 425 µm stretch of ventral and dorsal spinal cord. Student s two-tailed t- tests: Ventral spinal cord, average sheath number per cell, 9.3 +/- 3.6, control vs /- 4.2, PTZ, p = , n = 21 cells in 17 control and n = 43 cells in 30 PTZ treated animals; dorsal spinal cord, average sheath number per cell, 18 +/- 5.4, control vs /- 7, PTZ, p = 0.8, n = 11 cells in 10 control animals and n = 22 cells in 15 PTZ treated animals. (F) Oligodendrocytes labelled by Tg(mbp:EGFP) in control (top) and PTZ treated (bottom) animals. (G) Oligodendrocyte number in the ventral and dorsal spinal cord in control and PTZ treated animals in a 425µm stretch of spinal cord from somite level 8 to 11. Dorsal spinal cord, average oligodendrocyte number, /- 3, control vs /- 4, PTZ. Student s t-tests: ventral p = 0.006, dorsal p = 0.1, n = 48, control and n = 90 in PTZ. The reason for ventral spinal cord specific phenotypes lies in the fact that reticulospinal neurons, whose axons reside in the ventral spinal cord, receive inhibitory GABA-ergic input that is blocked by PTZ. However, Commissural Primary Ascending (CoPA) neurons, which we have recently identified as having myelinated axons, Mensch et al., in preparation, and that comprise many of the myelinated axons in the dorsal spinal cord do not receive such input, and so are not affected by PTZ.
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