Fig. 3.

KLF4 knockout increases RGC neurite growth in vitro and regeneration of adult RGCs in vivo. (A to C) Purified P12 RGCs were cultured from Thy1-cre^{−/ −}/KLF4^fl/fl/Rosa⁺ (Cre− WT) and Thy1-cre^+/−/KLF4^fl/fl/Rosa⁺ (Cre+ KO) mice, and plated for 3 DIV before Tau immunostaining and automated tracing. (A) Immunostaining for Tau (red) demonstrated low levels of growth of Cre− WT RGCs (left) but increased levels of axon growth of Cre+ KLF4-KO RGCs (right; Rosa+ yellow cells, arrowheads). (B) KLF4-KO RGCs have a higher percentage of cells with neurites, compared to controls (N = 3; *P < 0.02, t test; mean ± SEM). (C) When all YFP+ RGCs were measured, KLF4 KO RGCs extended longer neurites than WT RGCs (representative experiment shown; *P < 0.001; mean ± SEM). (D and E) Two weeks after optic nerve crush of Thy1-cre⁺/KLF4^+/+ (WT), Thy1-cre⁺/KLF4^fl/+ (Het), and Thy1-cre⁺/KLF4^fl/fl (KO) mice, regenerating fibers were anterogradely labeled by intravitreal injection of Alexa 594–labeled cholera toxin B. Regenerating fibers were counted at specified distances from the lesion site. (D) More fibers regenerate in KO mice compared to WT or Het (n = 10 WT, 4 Het, and 7 KO mice; P < 0.001 for KO versus WT or Het; no difference between WT and Het by mixed-model analysis of covariance; mean ± SEM). (E) Partial projections of sectioned optic nerve from WT and KO mice show regenerating axons more than 1 mm distal to the lesion site in KO nerve. Scale bar, 200 μm.