Figure 3.

A LARG/Rho/Rock pathway mediates C-RGMa inhibition: (a) In situ hybridization with a LARG anti-sense probe (LARG-AS) showed LARG expression by RGCs in the chick E8 retina. Negative control, LARG sense (LARG-S). Insets from the temporal and the nasal part of the retina show a low nasal, high temporal expression of LARG. (b) RGCs were nucleofected to express an RFP reporter together with either an miRNA or the PDZ domain for LARG. RGC axons that expressed the control pRFP plasmid appeared shorter when cultured on C-RGMa versus laminin. The expression of both miRNA for LARG or LARG-PDZ restored outgrowth on C-RGMa. (c) Quantification showed that LARG-PDZ and LARG miRNA significantly restored outgrowth on C-RGMa and not on N-RGMa. (d) PC12 cells treated with C-RGMa for 30 min showed a stronger signal for active Rho when compared with Control (BSA). (e) Quantifications show that C-RGMa significantly increased Rho activation (*P<0.005). (f) Temporal retinal explants were cultured on laminin+C-RGMa. Axonal inhibition by C-RGMa was suppressed by both the Rho inhibitor C3-transferase and the Rock inhibitor Y27632. (g) C3-transferase and Y27632 significantly reduced C-RGMa inhibition on RGC axons. Bars, 100μm