(A) Graphical representation of OBPI model. Boxes indicate injured and intact DREZ. (B) Confocal z-projection of Tg(ngn1:gfp) zebrafish at 4 dpf showing successful spinal root avulsion. (C) Representative images from 24-hour time-lapse movies starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish showing the arrival of both microglia and macrophages to the injury site. Arrows indicate microglia. Arrowheads indicate macrophages. Yellow box indicates injury site. (D) Confocal z-projection of Tg(pu1:gfp);Tg(sox10:mrfp) stained with the microglia-specific anti-4C4 antibody showing the presence of microglia in the brain and spinal cord and lack of 4C4 staining in macrophages outside the brain and spinal cord. Arrows indicate microglia. Arrowheads indicate macrophages. (E) Quantification of individual cell response time to site of injury. (F) Images from a 24-hour time-lapse movie starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish showing debris and vacuoles in phagocytic cells. Arrows indicate mRFP⁺ debris from sox10 cells. Arrowheads indicate vacuoles. (G) Quantification of the percentage of microglia and macrophages that contain debris (p < 0.0001). (H) Quantification of amount of individual debris puncta within microglia before and after arrival to injury site. Scale bar equals 1 μm (D,F), 10 μm (B,C). Statistics summarized in S1 Table. See S1 Data for raw data. DREZ, dorsal root entry zone; OBPI, obstetrical branchial plexus injury.

(A) Orthogonal rotation view of Tg(ngn1:gfp);Tg(sox10:mrfp) animals showing the proximity of the intact spinal root to the spinal cord. (B) Orthogonal rotation view of Tg(pu1:gfp);Tg(sox10:mrfp) animals showing microglia before PNS emigration, during emigration, and post-emigration back to the CNS. Arrows and green diagram color indicate microglia. Arrowheads and blue diagram color indicate macrophages. Dashed line indicates spinal cord boundary. (C) Quantification of the percentage of microglia that exit the CNS compared with those that remain in the CNS. (D) Quantification of the percentage of movies in which microglia exit the CNS at 4 dpf and 7 dpf. (E) Representative quantification of distance and time a microglia spent inside and outside of the CNS; y-axis > 0 indicates cell’s presence in PNS; y-axis < 0 indicates cell’s presence in CNS. (F) Images from a 24-hour time-lapse movie starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish showing microglia picking up debris while in the PNS and re-enter the CNS. Arrowheads indicate debris puncta while in CNS. Arrows indicate debris puncta while in the PNS. (G) Quantification of percentage of ectopic microglia that come back into CNS after leaving to the PNS compared with those that stay in PNS in a 24-hour imaging window. (H) Images from a 24-hour time-lapse movie starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish comparing the morphology of exiting and nonexiting microglia. Red arrows indicate projections created by microglia. (I) Shape description quantification of the average aspect ratio of microglia in the CNS, in the PNS, and PNS-primed in the CNS (p = 0.0010; p = 0.0015; p = 0.6353). (J) Shape description quantification of the average roundness of microglia in the CNS, in the PNS, and PNS-primed in the CNS (p < 0.0001; p < 0.0001; p = 0.4689). (K) Quantification of the total amount of new debris collected by microglia (p = 0.0319; p = 0.5370; p = 0.0408). (L) Stitched zoning images from a 24-hour time-lapse movie starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish showing the entire animal. White boxes coordinated with letter tags represented zones of the animal in which microglia traveled; larger images located below. Yellow box indicates injury site. Arrows indicate microglia. Arrowheads indicate macrophages. Scale bar equals 1 μm (F,H), 10 μm (bottom L), and 100 μm (top L). Statistics summarized in S1 Table. See S2 Data for raw data. CNS, central nervous system; dpf, days post fertilization; PNS, peripheral nervous system.

(A) Quantification of the average number of new debris from PNS-primed microglia collected at the secondary injury site compared to naïve CNS microglia (p = 0.0273). (B) Quantification of the percentage of movies in which microglia exit the CNS in DMSO, NMDA inhibitor treated, and glutamate uncaged in PNS cases. (C) Images from 24-hour time-lapse movies starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) MNI-L-glutamate treated zebrafish showing the response of microglia pre- and post-mock glutamate uncaging. Arrowheads denote microglia. Yellow box indicates uncaging site. (D) Images from 24-hour time-lapse movies starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) MNI-L-glutamate treated zebrafish showing the response and ectopic migration of microglia pre- and post-glutamate uncaging. White arrow denotes first microglia; blue arrow indicates a second microglia. Yellow box indicates uncaging site. (E) Quantification of the average time microglia took to exit the CNS post-glutamate uncaging (p = 0.0379). Scale bar equals 10 μm (C, D). Statistics summarized in S1 Table. See S3 Data for raw data. CNS, central nervous system; dpf, days post fertilization; MNI-L, 4-Methoxy-7-nitroindolinyl-caged-L-glutamate; NMDA, N-methyl-D-aspartate receptor; PNS, peripheral nervous system.

(A) Images from 24-hour time-lapse movies starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish showing points of cell–cell contact. Blue arrowheads indicate microglia. Green arrowheads indicate macrophages. Yellow box indicates contact. (B) Quantification of distance traveled pre- and post-contact between a microglia and macrophage. (C) Quantification of percentage of cells experiencing directional change post-contact with migrating cell (p = 0.0164; p = 0.0033). (D) Images from a 24-hour time-lapse movie starting at 4 dpf in Tg(pu1:gfp);Tg(sox10:mrfp) zebrafish showing microglia extending a cellular process into the periphery. Dashed line indicates sox10⁺ boundary. Yellow circle indicates tip of cellular projection. (E) Images from a time-lapse ablation window in Tg(pu1:gfp) zebrafish at 4 dpf showing successful ablation of macrophage and immediate microglial response. Green dashed line indicates spinal cord boundary. Bracket indicates distance between microglia and ablated macrophage. (F) Quantification of average time microglia spent responding to injury. (G) Quantification of average distance microglia traveled to the macrophage or control ablation site (p = 0.0462). (H) Quantification of percentage of injuries containing sox10⁺ puncta. (I) Quantification of number of free-roaming debris puncta compared to puncta localized within cells. Scale bar equals 10 μm (A, D, and E). Statistics summarized in S1 Table. See S4 Data for raw data. dpf, days post fertilization.