Morphogenetic defects after morpholino-mediated downregulation of rtn4a and rtn4b. (A) At 15 hpf, control embryos showed a differentiated eye anlage (e) and forebrain (arrow). In contrast, the general morphology of rtn4a- and rtn4b-morpholino (MO)-injected embryos was visibly affected (D) to (G). The forebrain was flattened (arrow), and the eye anlage (e) and the head were reduced in size. At 1 dpf, the heads and eyes of embryos injected with rtn4a and rtn4b MO1 remained reduced. In particular, rtn4b morphants exhibited an abnormally curved notochord (E) to (H). At 2 dpf, the rtn4a morphants still had reduced eyes and forebrains compared to controls (Co) (F and F2 vs. C and C2), but no other morphogenetic defects were apparent. Rtn4b morphants had even smaller eyes, markedly shortened forebrain/midbrain regions and a deformed fourth ventricle (I and I2, arrow). At 3 dpf, the eyes and brains of rtn4a and rtn4b morphants remained smaller (J, L and N). Rtn4b morphants developed a thinner, ventrally curved tail; lacked lower jaws (asterisk); and had an inflated heart cavity. At 5 dpf, rtn4a, but not rtn4b, morphants seemed to regain a nearly normal overall morphology (K, M and O). The reduction in eye size was quantified at 15 hpf in embryos injected with rtn4b MO1 and MO2 (targeting the 52 untranslated region and ATG, respectively) and coinjected with rtn4b mRNA (P). In rescue experiments, the rtn4b morphant phenotype showed clear improvement (Q). Anterior brain structures selected for measurements are outlined in yellow. Samples studied were rtn4b MO1 (n = 36), rtn4b MO2 (n = 64), rtn4b MO1 + rtn4b mRNA (n = 69) and rtn4b MO1 (n = 85). Scale bars = 100 μm.