Lh3 in sox10-expressing glia restores RGC axonal growth across the midline. (A) In wild-type zebrafish larvae expressing Tg(isl2b:GFP) to label RGCs, macrophages and microglia labeled by Tg(mpeg1:Gal4FF);Tg(UAS:nfsB-mCherry) are observed in the area of the optic chiasm before transection. At 24 h post-transection (hpt), an influx of mpeg+ cells is seen. (B) Quantification of the percentage of nerves at 72 hpt that grew across the midline to re-innervate the optic tectum, did not grow across the midline and instead grew along aberrant trajectories, or showed stalled axonal growth with and without the Tg(mpeg:lh3-myc) transgene. Numbers indicate total number of transected nerves. ****P<0.0001; n.s., P>0.05 (Fisher's exact test). (C,D) Oligodendrocyte lineage cells labeled by Tg(olig2:dsred) as well as mbpa mRNA expression in myelinating oligodendrocytes are also seen in the chiasm before and after transection, near the injured proximal stump of RGC axons or along the degenerating original projection. (E) Quantification of regeneration with and without Tg(sox10:lh3-mkate) transgene. Numbers indicate total number of transected nerves. ****P<0.0001; n.s., P>0.05 (Fisher's exact test). (F) lh3 mRNA expression at 4-6 hpt is upregulated and overlaps with oligodendrocytes labeled by Tg(olig2:dsred). Scale bar: 50 μm (bar in F applies to A,C,D,F). (G) Additional images of individual oligodendrocytes (arrowheads) labeled by Tg(olig2:dsred) co-expressing lh3 mRNA (see also Movie 3). Scale bar: 10 μm. Representative images across timepoints are of different larvae. Axons in the chiasm region at 4-6 hpt have not yet fully degenerated following transection. Dashed lines indicate the outline of the eyes.
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