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. 2019 Aug 16;9:11945. doi: 10.1038/s41598-019-48421-9

Figure 5.

Figure 5

Spinal motor axon growth cones require HD-PTP for ephrin-B2-induced collapse. (a) Representative images of GFP+ growth cones from dissociated ControlCRISPR- or HD-PTPCRISPR-electroporated motor neurons, incubated with eB2, Sema3F, or Fc, and stained with anti-GFP antibodies. (b) Representative images of growth cones from rescue experiments using dissociated motor neurons electroporated with ControlCRISPR plasmid or HD-PTPCRISPR co-electroporated with hHD-PTP or hHD-PTP C/S plasmid, incubated 30 min with 10 µg/mL eB2 or Fc and stained with anti-HD-PTP antibodies. (c) Quantification of collapsed growth cones in dissociated motor neurons electroporated with CRISPR constructs and stimulated with ligands. HD-PTPCRISPR or ControlCRISPR growth cones were incubated for 30 min with 10 µg/mL eB2 or Fc. The collapse response of HD-PTPCRISPR growth cones to eB2 was significantly attenuated compared to ControlCRISPR (n = 3, 90 growth cones/n; p < 0.0001; Fisher’s exact test). Rescue experiments with growth cones from dissociated motor neurons electroporated with ControlCRISPR, or HD-PTPCRISPR co-electroporated with hHD-PTP or hHD-PTP C/S expression plasmid, incubated 30 min with 10 µg/mL eB2 or Fc and stained with anti-HD-PTP and anti-Isl1 antibodies. Both populations responded to eB2 treatment indistinguishably from control (n = 4, 50 growth cones/n; Fisher’s exact test). HD-PTPCRISPR or ControlCRISPR growth cones were incubated for 30 min with 0.3 µg/mL Sema3F-Fc or Fc. The two CRISPR growth cone populations behaved identically, demonstrating that HD-PTP loss does not affect the response to Sema3F (n = 4, 30 growth cones/n; Fisher’s exact test). Values are plotted as mean ± SD. All values can be found in Supplementary Table S4. h: human; S3F: Sema3F; eB2: ephrin-B2-Fc; ***p < 0.001; n.s.: not significant. Inverted grayscale fluorescent images.