Slit2 is necessary for optic axon organization in the zebrafish ventral midline

The functional connection of the retina with the brain implies the extension of retinal ganglion cells axons through a long and tortuous path. Slit-Robo signaling has been implicated in several steps of this journey, with a central role assigned to Slit2. We report here the phenotypical characterization of a zebrafish null mutant for this gene, generated by CRISPR-Cas9 technology, along with a detailed analysis of its expression pattern by WM-FISH. All evident defects in the optic axons in slit2-/- mutants were detected outside the retina, coincident with the major sites of expression at the ventral forebrain, around the developing optic nerve and anterior to the optic chiasm/proximal tract. Anterograde axon tracing experiments in zygotic and maternal-zygotic mutants, as well as morphants, showed the occurrence of different axon sorting defects, which appeared mild at the optic nerve level, but more severe in the optic chiasm and the proximal tract. A remarkable phenotypical feature was the usual splitting of one of the optic nerves in two branches that surrounded the contralateral nerve at the chiasm. Although all axons eventually crossed the midline, the retinotopic order appeared lost at the proximal optic tract, to eventually correct distally. A detailed time-lapse analysis demonstrated the sporadic occurrence of axon misrouting at the chiasm level, which could be responsible for the sorting errors. Our results support previous evidence of a channeling role for Slit molecules in retinal ganglion cell axons at the optic nerve level, in addition to a function in the segregation of axons coming from each nerve and from different retinal regions at the medio-ventral area of the forebrain.