Figure 2

Guidance of retinal axons on an EphA7-Fc versus Fc substrate is abolished by knockdown of p75^NTR. (A) Efficiency of selected RNAi experiments to knock down p75^NTR protein. Chick or rat p75^{NTR, FLAG} was expressed in CHO cells in parallel to different RNAi experiments targeting selected chick p75^NTR sequences. RNAi(2) almost completely knocks down chick but not rat p75^NTR, while RNAi(3) has no effect on either. α-Tubulin levels are shown for loading controls. (B) Single cells from embryonic day 6 nasal thirds of chick retina were electroporated with RNAi vectors for p75^NTR. These RNAi vectors also contained a red fluorescent protein (RFP) cassette. Then cells were plated on a substrate of alternating lanes of EphA7-Fc and Fc stripes. The first generated stripe was labelled by adding FITC-Fc at low concentrations (indicated by EphA7-Fc*, or Fc* in Fc/Fc controls). In the pictures shown, the green stripes represent the EphA7-Fc stripe. Stripe width is 50 μm. Two days later, outgrowth preferences of retinal axons were analysed. The picture on the left shows the growth preference on EphA7-Fc*/Fc stripes after electroporation of control RNAi(3), and the picture on the right that after electroporation of RNAi(2). Knockdown of p75^NTR leads to a strong abolishment of the striped outgrowth of retinal axons. For all stripe assays (and outgrowth/branching assays) the evaluation was done blind to the composition of stripes, constructs transfected and ligands added. (C) Quantification of growth preferences after p75^NTR knockdown. The results of three independent experiments are shown. Error bars denote standard error of the mean and significance is indicated as ***P < 0.001; n.s., not significant. Statistical analysis was done in GraphPad using one way ANOVA and Tukey post hoc test (GraphPad: LaJolla, CA, USA).