Dorsal root ganglion (DRG) sensory neurons are ineffective at regrowing central branch axons following axotomy. (a) Diagram of a larval zebrafish DRG at 3 days postfertilization (dpf) with central branch axotomy (CBA) laser ablation paradigm (D, dorsal; A, anterior). (b) In vivo, time-lapse imaging of a Tg(ngn1:gfp;sox10:nls-eos) DRG at 3 dpf before (Pre-injury) and post-CBA (?0 hours post-injury [hpi]) with fluorescently labeled sensory neuron (asterisk; neurite tip indicated by arrow), satellite glial cell nuclei (white arrowheads), and SC nuclei (open arrowhead). Spinal cord boundary indicated by dashed line. (c) Percent of DRGs with successful or unsuccessful central branch regrowth post-CBA (n = 39 DRGs from 22 fish). (d) Number of new dorsal neurite outgrowths per DRG post-CBA. (e) Quantification of time post-CBA at which the first dorsal neurite forms and initiates growth from the sensory neuronal soma (n = 16 neurites from 11 fish; dashed line, mean: 2.77 ± 1.66 h post-CBA). (f) Survival curves of primary (solid line) and secondary (dashed line) neurite longevity. Vertical lines mark the time (h) at which neurite survival probability is 50% (n = 30 neurites from 11 fish; mean: 4.91 ± 5.15 h; log-rank test, nsp >.05). Scale bar: 10 ?m.

Satellite glia respond to central branch axotomy (CBA) by relocating their nuclei dorsally toward the injury site. (a) Diagram of trunk dorsal root ganglion (DRG) CBA injury observations over time (hours post-injury [hpi]). Post-CBA (?CBA?), neurons may grow new dorsal neurites in an attempt to regrow a central branch axon. During this period, a subset of satellite glia (SGC) nuclei move dorsally. (b) Representative migration paths over time of SGC nuclei from uninjured (Control, gray circles) and injured (Post-CBA, rose diamonds) DRGs, relative to the neuronal soma (asterisk). Larger, outlined symbols indicate initial (lighter color) and end (darker color) positions for each cell. (c) Graph of SGC nuclear displacement from initial positions at end of in vivo, time-lapse imaging, comparing SGCs from uninjured (mean: 1.00 ± 1.09 a.u.) and sham (mean: 0.83 ± 1.11 a.u.) injury control (?CON?) DRGs (n = 28 cells from 10 DRGs from 5 fish; Wilcoxon test, p > .05). Dashed line indicates mean; measures normalized to uninjured controls. (d?f) Graphs comparing SGC nuclear displacement (mean: control (CON) 1.00 ± 1.09 a.u., CBA 1.43 ± 0.84 a.u.; Wilcoxon test, p = .038), speed (mean: CON 1.00 ± 0.25 a.u., CBA 1.31 ± 0.18 a.u.; Wilcoxon test, p < .001), and velocity (mean: CON 1.00 ± 1.08 a.u., CBA 1.66 ± 0.87 a.u.; Wilcoxon test, p = .019). Dashed line indicates mean (n = 64 cells from 27 DRGs from 19 fish); measures normalized to CON. Triangles indicate SGC nuclei from DRGs with successful regrowth of the central branch post-CBA and circles represent SGC nuclei from DRGs that did not successfully regrow the central branch. (g) Density distribution graphs of SGC nucleus initial and end positions (relative to the neuronal soma) from CON and CBA DRGs. Arrows represent net change in SGC X- (mean: CON 1.64 ± 2.25 ?m, CBA 1.36 ± 3.29 ?m; Wilcoxon test, p = .26) and Y-positions (mean: CON ?0.16 ± 4.22 ?m, CBA 3.10 ± 3.68 ?m; Wilcoxon test, p = .00031) from initial and end timepoints each condition. (h) Circular plots of SGC nucleus angular position relative to the neuronal soma at initial and end timepoints from CON (mean: Initial 156 ± 125°, end 27 ± 108°; Watson's U2 two-sample test, p > .05) and CBA (mean: Initial 204 ± 117°, end 82 ± 78°; Watson's U2 two-sample test, p < .05) conditions. Dashed arrows indicate mean initial angular position, solid arrows indicate mean end angular position, shaded regions indicate ±1 standard deviation, and star bursts indicate mean central axon angular position pre-injury. nsp ?.05, *p < .05, **p ? .01, ***p ? .001.

Satellite glial cell ablation with central branch ablation improves central branch regrowth rate. (a) Diagram of dorsal root ganglion (DRG) central branch ablation (CBA) and satellite glia ablation (SGC-Abl) with CBA laser injury paradigms. (b) Percent of DRGs with successful and unsuccessful central branch regrowth into the dorsal spinal cord following CBA (n = 5/28 DRGs from 18 fish) with or without SGC-Abl (n = 9/17 DRGs from 14 fish; Pearson's ?² test, p = .033). (c) In vivo, time-lapse imaging of a Tg(ngn1:gfp;sox10:nls-eos) DRG at 3 dpf before (pre-injury) and after SGC-Abl CBA (?0 h postfertilization, hpf) with fluorescently labeled sensory neuron (asterisk; new dorsal neurite indicated by arrow) and SGC nuclei (white arrowheads). Spinal cord boundary indicated by dashed line. Single Z-plane inset indicated by square outline. Scale bars: 10 ?m. *p < .05.

Dorsal root ganglion sensory neurons are more likely to regrow a new central branch when subject to satellite glial cell (SGC) ablation. (a) Comparison of the number of dorsal root ganglia (DRG) that exhibit either zero or one or more dorsal neurite outgrowths following CBA (light fill, 61% with one or more dorsal neurite outgrowths; n = 17/28 DRGs from 18 fish) or (SGC-ABL) with CBA (dark fill, 88% with one or more dorsal neurite outgrowths; n = 15/17 DRGs from 14 fish; Barnard's test, p = .038). (b) Survival curves of neurite longevity from CBA (light line, mean: 2.89 ± 3.59 h) and SGC-Abl CBA (dark line, mean: 3.21 ± 4.49 h) injury conditions. Vertical lines mark the time (h) at which neurite survival probability is 50% (n = 37 neurites from 21 DRGs from 15 fish; log-rank test, p > .05). (c?e) Quantification of time (hours post-injury [hpi]) at which the first dorsal neurite forms and initiates growth from the sensory neuronal soma following (c) CBA alone (light, mean: 3.38 ± 3.74 hpi) or (d) SGC-Abl CBA (dark, mean: 2.14 ± 2.31 hpi). Curves represent smoothed density estimates. Dashed lines indicate mean initiation time for each condition (n = 31 DRG primary neurites from 19 fish; Wilcoxon test, p > .05). (f) Quantification of initiation time of neurites that fail (light curve, mean: 5.61 ± 4.59 hpi) or successfully regrow into the dorsal spinal cord (dark curve, mean: 2.64 ± 3.70 hpi), pooled from both injury conditions (n = 52 neurites from 30 DRGs from 19 fish; Wilcoxon test, p = .0054). Dashed lines indicate mean initiation time. (g) Quantification of the percent of neurites present in 1 h time windows post-injury, compared between CBA (2 hpi mean: 16% ± 2%, 4.5/28 neurites) and SGC-Abl CBA neurons (2 hpi mean: 45% ± 8%, 10.75/24 neurites; Wilcoxon test with Bonferroni's correction, p < .05). (h) Mean displacement rate (?m/imaging time interval) of SGC nuclei in 1 h time windows post-CBA (n = 43 cells from 23 DRGs from 15 fish; Wilcoxon paired-sample test with Bonferroni's correction, p < .05). Error bars represent ±1 standard deviation. nsp ?.05, *p < .05.

ErbB signaling inhibition improves central branch regrowth. (a) Diagram of ErbBi central branch axotomy (CBA) ablation paradigm. Larval zebrafish were preincubated with 4 ?m ErbB inhibitor AG-1478 in 1% DMSO (ErbBi CBA) or vehicle control (DMSO CBA). (b) Percent of dorsal root ganglia (DRG) with successful or unsuccessful central branch regrowth into the dorsal spinal cord following CBA with DMSO or ErbBi (n = 32 DRGs from 24 fish; Pearson's ?² test, p = .015). (c) In vivo, time-lapse imaging of a Tg(ngn1:gfp;sox10:nls-eos) DRG at 3 dpf under ErbBi conditions before (Pre-injury) and after CBA (?0 h post-injury, hpi) with fluorescently labeled sensory neuron (asterisk; new dorsal neurite indicated by arrow) and satellite glial cell SGC nuclei (white arrowheads). Spinal cord boundary indicated by dashed line. Single Z-plane inset indicated by square outline. (d) Comparison of the number of DRGs that exhibit either zero or one or more dorsal neurite outgrowths following CBA, under DMSO (white fill, 35% with one or more dorsal neurite outgrowths; n = 6/17 DRGs from 13 fish) or ErbBi (dark fill, 86% with one or more dorsal neurite outgrowths; n = 12/14 DRGs from 11 fish; Barnard's test, p = .0047) conditions. (e) Quantification of initiation time (hours post-injury [hpi]) of neurites that fail (light curve, mean: 3.32 ± 2.87 hpi) or successfully regrow into the dorsal spinal cord (dark curve, mean: 1.25 ± 2.45 hpi), pooled from both DMSO and ErbBi treatment conditions (n = 25 neurites from 19 DRGs from 13 fish; Wilcoxon test, p = .023). Dashed lines indicate mean initiation time. (f) Graph comparing SGC nuclear speed under DMSO (mean: 1.00 ± 0.27 a.u.) and ErbBi (mean: 0.85 ± 0.30 a.u.) treatment conditions (n = 68 cells from 29 DRGs from 19 fish; Wilcoxon test, p = .0040). Dashed line indicates mean; measures normalized to DMSO. Triangles indicate SGC nuclei from DRGs with successful regrowth of the central branch and circles represent SGC nuclei from DRGs that did not successfully regrow the central branch. (g) Circular plots of SGC nucleus angular position relative to the neuronal soma at initial and end timepoints from the ErBbi CBA (mean: Initial 297 ± 87°, End 337 ± 92°; Watson's U2 two-sample test, p > .05) condition. Dashed arrows indicate mean initial angular position, solid arrows indicate mean end angular position, shaded regions indicate ±1standard deviation, and star bursts indicate mean central axon angular position pre-injury. Scale bars: 10 ?m. nsp ?.05, *p < .05, **p ? .01