The CPEB1 mutant (S174A, S180A) (CPEB1-AA) disrupts axon outgrowth in vivo. (A) Retinal ganglion cells (RGCs) transfected with CPEB1-RBM-GFP (RBM) send axons to the optic tectum correctly. ch, optic chiasm. (B) Embryos where RGCs are transfected with CPEB1-AA-GFP (AA) do not have GFP-positive axons in the brain. (C) Schematic of bidirectional plasmid encoding both GAP-red fluorescent protein (RFP) and CPEB1-green fluorescent protein (GFP). CMV, cytomegalovirus; mPGK, mouse phosphoglycerate kinase. (D-G) Electroporation of bidirectional plasmids causes co-expression of GAP-RFP and CPEB1-GFP. Dashed boxes in (D) and (F) are shown at higher resolution in (E) and (G), respectively. (H) Diagram of optic pathway in horizontal sections. Dashed boxes indicate the part of the pathway shown in (I), (J), and (K). (I) RFP/RBM- and RFP/AA-transfected axons in the optic nerve head. These axons contain CPEB1-GFP (arrows). (J) Very rarely, an RFP/AA-transfected axon extends just beyond the optic chiasm. Dashed lines indicate expected path of retinal axons. (K) RFP/RBM-transfected, but not RFP/AA-transfected, axons reach and branch in the tectum (arrowhead). The RFP/RBM image is a composite of two adjacent sections. (L) Number of RFP/RBM- and RFP/AA-transfected embryos with detectable RFP-positive axons reaching each intermediate target, analyzed in sections. (M-N) RFP/RBM-transfected embryos have bright RFP-positive axons in the optic pathway projecting correctly to the tectum, while RFP/AA-transfected embryos do not. Scale bars: 65 μm (A, B, M, N); 10 μm (D, F, J, K); 5 μm (I). Blue, DAPI; green, CPEB1-GFP; red, GAP-RFP. Dashed lines indicate inner and outer plexiform layers and solid lines indicate the apical and basal limits of the retina. Abbreviations: INL, inner nuclear layer; ONL, outer nuclear layer.