Figure 5: gpr56 mutant OPCs form premature associations with axons and are less proliferative than controls.

(a,b) Representative stills from in vivo time-lapse imaging of the spinal cord (white bracket at 16 h) from segments 5 to 7 of (a) controls (gpr56^stl13/+, N=6) and (b) gpr56^stl13/stl13 mutants (N=7) expressing tg(sox10(−7.2):mRFP) to visualize OPC behaviour over time (three technical replicates performed). Embryos were imaged from 30 to 46 hpf to encompass the transition of OPCs (white arrowheads) into pro-OLs (light green arrowheads). White arrows denote sox10 expressing neural crest cells, and orange arrows mark the posterior lateral line nerve, which is part of the peripheral nervous system and is marked by the presence of migrating Schwann cell precursors. (c) Quantification of the number of pro-OL associated axons over time in gpr56 mutants compared with controls at 30 hpf (P<0.039), 38 hpf (P<0.28) and 46 hpf (P=0.004). (d–f) Representative TEM images of the ventral spinal cord of a WT (d, N=3) and gpr56^stl13/stl13 (e,f, N=4) embryo at 3 dpf (two technical replicates performed). (e) Additional non-Mauthner myelinated axons (shaded blue and numbered) were observed in gpr56^stl13/stl13 embryos (P<0.048) compared with controls. Mauthner axon shaded orange. (f) Image shows a higher magnification of panel e (green box). (g) Raw counts of non-Mauthner myelinated axons in control and gpr56^stl13/stl13 embryos at 3 dpf. (h,i) Representative fluorescent images of the spinal cord from segments 5 to 7 of (h) control (gpr56^stl13/+, N=31) and (i) gpr56^stl13/stl13 mutant (N=34) embryos at 32 hpf (two technical replicates performed). Embryos expressing tg(sox10 (−7.2):mRFP) were fixed and stained with pH3 to assay proliferation. (j) Quantification of the number of proliferating OPCs in gpr56^stl13/stl13 mutants compared with controls (P<0.0004). (k–m) Quantification of dying cells (acridine orange +, one technical replicate per time point) in gpr56^stl13/stl13 mutants and controls (WT and gpr56^stl13/+) at 32 hpf (control: N=13, gpr56^stl13/stl13: N=20), 2 dpf (control: N=21, gpr56^stl13/stl13: N=12) and 3 dpf (control: N=19, gpr56^stl13/stl13: N=20). (a,b,h,i) Scale bar, 50 μm. (d,e) Scale bar, 1 μm. (f) Scale bar, 500 nm. Student’s t-test used to test for statistical significance and error bars shown as±s.d.