Co-expression and onset analysis of amacrine and horizontal markers Ptf1a and AP2α. (A) Developing horizontal or amacrine neurons do not express Brn3b. Sections from E13.5 retinas coimmunostained for Brn3b, AP2α and Ptf1a. Although many Brn3b+ cells are present, none co-label with the horizontal and inhibitory amacrine marker Ptf1a, or the pan-amacrine marker AP2α, suggesting that Brn3b specifically marks RGCs during early development. (B–E) Sections from E13.5 (B,D) or E15.5 (C,E) embryos co-stained with Ptf1a, AP2α and EdU following a 4 hr (B,C) or 12 hr (D,E) chase. No Ptf1a+ or AP2α+ cells are EdU+ after a 4 hr chase, suggesting that these factors are expressed exclusively in post-mitotic (G0) cells. Many Ptf1a+ EdU+ cells are apparent after a 12 hr chase at both E13.5 and E15.5 (arrows, insets), indicating that this factor initiates expression in migratory cells. Many AP2α+ cells are Ptf1a+ (arrowheads), marking the inhibitory amacrine population. Scale bar, 50 μm.