Regenerating axons navigate a non-neural lesion environment that is rich in ECM independently of astroglia-like processes. a Time-lapse video-microscopy reveals that axonal growth cones (arrow, Xla.Tubb:DsRED) extend into the lesion site independently of astroglia-like processes (her4.3:EGFP, empty arrow points out lack of glial labelling). Arrowhead indicates glial process extending into the lesion site. Single frames are shown. Abbreviations: fin, dorsal fin fold; sc, spinal cord; nc, notochord. b Quantification of fascicle composition at 1 dpl using different combinations of immunohistochemical and transgenic markers for astroglia-like processes and axons indicates that the majority of fascicles are neuronal with no detectable glial component (46–62% of all fascicles analyzed). In the example scans on the left, yellow arrows indicate mixed fascicles, containing neurites and glial processes; green arrows indicate pure glial fascicles and red arrows indicate pure axonal fascicles. c Time course of re-growth of axons (anti-acetylated Tubulin+) and astroglia-like processes (anti-GFAP+) after spinal cord transection. Quantification of labelling continuity between rostral and caudal spinal cord stumps at 1 dpl and 2 dpl suggests faster bridging of the lesion site by neuronal rather than glial processes. Arrow points to axonal bridge with very few glial processes in the lesion site at 1 dpl at higher magnification (Fischer’s exact test: **P < 0.01; n.s. indicates not significant). d Treatment of gfap:Gal4ff;UAS:NTR-mCherry transgenic fish with the pro-drug Metronidozole (MTZ) almost completely ablates transgene-expressing glial cells by 3 dpf. e Treatment of gfap:Gal4ff;UAS:NTR-mCherry transgenic fish with MTZ dramatically reduces anti-GFAP+astroglia-like processes (t-test: ***P < 0.001) but does not reduce the proportion of larvae with axonal bridges at 2 dpl (Fischer’s exact test: n.s indicates not significant). f fn1a and col1a2 expression is upregulated after lesion, shown by in situ hybridization. g Regenerating axons (anti-acetylated Tubulin+; arrows) are closely associated with Collagen I and Fibronectin immunoreactivity in a lesion site (confocal depth was limited to spinal cord). Also see Supplementary Movies 3 and 4. a–g Views are dorsal (a; rostral is left) or lateral (b-g; dorsal is up, rostral is left). BF: brightfield. Scale bars: 100 µm a,c–f and 50 µm b,g. Error bars indicate s.e.m
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