Mst3b is essential for optic nerve regeneration. RGCs were infected with AAV2 expressing EGFP plus either control or mst3b shRNA. Four weeks later, the optic nerve was crushed and the lens was injured to induce inflammation and transform RGCs into an active growth state. Regeneration was quantified 2 weeks later. (a) Longitudinal sections through the optic nerve stained for GAP-43 (red, a marker for regenerating axons) and EGFP (green, a marker for transfected neurons). Asterisk denotes the site of nerve injury; box shows the region magnified in b. Scale bar: 100 μm. (b) Area distal to the injury site from a case expressing mst3b shRNA. Merged image shows that GAP-43-positive axons that arise from uninfected RGCs (red), but not double-labeled axons (yellow) that arise from transfected neurons, extend distal to the injury site. Arrows indicate regenerating axons from uninfected RGCs. Scale bar: 100 μm. (c) Mean number of axons that regenerate ≥ 500 μm beyond the injury site. The set of bars on the left shows axons that arise from transfected RGCs. The number of regenerating axons was normalized by the percentage of transfected cells to account for differences in infection efficiency. The set of bars on the right shows axons of noninfected cells, normalized by the percentage of noninfected cells. Results are based on 7 animals per group, 4-6 sections per animal, and 0-24 axons per section. †††decrease relative to cases transfected with control shRNA significant at P < 0.001. Error bars represent s.e.m.