Brain differentiation and retinotectal projections in rtn4a- and rtn4b-knockdown, GFP–transgenic embryos. In rtn4a-morpholino (MO)- and rtn4b-MO-injected embryos, brains are smaller both anteroposteriorly as well as laterally. (A) Extent of the tectum (t) and the forebrain (fb) marked by white brackets in the HuC/HuD labeled brains of control embryos. (D) and (G) Knockdown of rtn4a, but more severely of rtn4b, leads to a reduction in the size of the tectum and the forebrain. In rtn4b morphants, the olfactory placodes (arrowhead) are not clearly identifiable and the tectum is localized in abnormally anterior positions. (B) In control embryos, the GFP–labeled retinal ganglion cell (RGC) axons cover the tectal neuropil (outlined). (E) and (H) The RGC axons in the tectum cover a smaller area in both morphants, but more severely so in the rtn4b morphants than in their rtn4a counterparts. GFP is also expressed in neuromasts (arrows), which are aberrantly positioned in the morphants. (C), (F) and (I) 42,6-diamidino-2-phenylindole (DAPI) staining of the brain shows the area of the neuropil (outlined), which is reduced after downregulation of rtn4a and rtn4b. (J), (K) and (L) Quantification of the length of the optic tectum, forebrain and tectal neuropil in control and rtn4a-MO- and rtn4b-MO-injected embryos. (A) to (I) Dorsal views of the brain of tg(brn3c:mGFP) embryos at 5 dpf. Control MO (5.0 ng) (n = 10), rtn4a-l-MO1 (5.0 ng) (n = 10) and rtn4b-MO1 (5.0 ng) (n = 10). Scale bar = 100 μm.