Development of neural and non-neuronal structures in rtn4b-knockdown, GFP transgenic embryos. At 1 dpf, tg(Shh:GFP), rtn4b morphant embryos showed deformations in the hypothalamic region (h), dorsal thalamic structures (arrow), anterior floor plate (f) and forebrain (A) and (B). Additionally, the notochord (n) is abnormally undulated (compare (C) and (D)). In addition, 1-dpf tg(hb9:GFP), rtn4b morphant embryos had fewer cell bodies in the spinal cord and truncated motor axons (E) and (F) (arrowheads). At 2 dpf, motor axons showed aberrant branching (G) and (H) (arrows). At 5 dpf, lateral (J) and dorsal (L) views reveal smaller eyes (e), aberrant retinal ganglion cells (RGCs) (white arrow) and few retinal axons projecting into the tectum (t), which is displaced anteriorly, along with abnormally patterned branchial arches (br) and reduced pectoral fins (pf) (I) to (L). At 2 dpf, tg(Isl1:gfp), rtn4b-MO1 morphants showed severe defects in cranial motor neurons, including lack of pairs III and IV (M) and (N). (O) Quantification of the number of motor neurons in posterior segments (n = 15 to 20) of the spinal cord in control morpholino (Co MO; n = 20), rtn4b-MO1 (n = 25) and rescued (n = 20) embryos at 1 dpf. (P) Proportion of embryos with abnormal motor axons in embryos injected with Co MO, rtn4b-MO, rtn4b-MO2, or coinjected with half concentrations of both MOs, or rtn4b-MO1 + rtn4b-mRNA, and injected with rtn4b-mRNA. hb: hindbrain; e: eye; ob: olfactory bulb; c: cerebellum; X: branchiomeric nerve X. Co MO (n = 15), rtn4b-MO1 (2.5 ng) (n = 30), rtn4b-MO2 (2.5 ng) (n = 31), rtn4b-MO1 (5.0 ng) (n = 17), rtn4b-MO2 (5.0 ng) (n = 11), rtn4b-MO1 + rtn4b-MO2 (2.5 ng) (n = 20), rtn4b-MO1 (5.0 ng) + rtn4b-mRNA (100 pg) (n = 69) and rtn4b mRNA (100 pg) (n = 85). Scale bar = 100 μm.