Background: As a consequence of gene/genome duplication, the RTN4/Nogo gene has two counterparts in zebrafish: rtn4a and rtn4b. The shared presence of four specific amino acid motifs-M1 to M4-in the N-terminal region of mammalian RTN4, and zebrafish Rtn4b suggests that Rtn4b is the closest homologue of mammalian Nogo-A.Results: To explore their combined roles in zebrafish development, we characterized the expression patterns of rtn4a and rtn4b in a comparative manner and performed morpholino-mediated knockdowns. Although both genes were coexpressed in the neural tube and developing brain at early stages, they progressively acquired distinct expression domains such as the spinal cord (rtn4b) and somites (rtn4a). Downregulation of rtn4a and rtn4b caused severe brain abnormalities, with rtn4b knockdown severely affecting the spinal cord and leading to immobility. In addition, the retinotectal projection was severely affected in both morphants, as the retina and optic tectum appeared smaller and only few retinal axons reached the abnormally reduced tectal neuropil. The neuronal defects were more persistent in rtn4b morphants. Moreover, the latter often lacked pectoral fins and lower jaws and had malformed branchial arches. Notably, these defects led to larval death in rtn4b, but not in rtn4a morphants.Conclusions: In contrast to mammalian Nogo-A, its zebrafish homologues, rtn4a and particularly rtn4b, are essential for embryonic development and patterning of the nervous system.