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

Abstract

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 axon growth and guidance in several steps of this journey. Here, we analyzed in detail the expression pattern of slit2 in zebrafish embryos by whole-mount fluorescent in situ hybridization, to extend previous work on this and other species. Major sites of expression are amacrine cells in the retina from 40 hpf, as well as earlier expression around the future optic nerve, anterior to the optic chiasm, two prominent cell groups in the anterior forebrain and the ventral midline of the caudal brain and spinal cord. To further characterize slit2 function in retinal axon growth and guidance, we generated and phenotypically characterized a null mutant for this gene, using CRISPR-Cas9 technology. Although no evident defects were found on intraretinal axon growth or in the formation of the optic tracts or tectal innervation, we observed very characteristic and robust impairment on axon fasciculation at the optic nerves and chiasm. The optic nerves appeared thicker and defasciculated only in maternal-zygotic mutants, while a very particular unilateral nerve-splitting phenotype was evident at the optic chiasm in a good proportion of both zygotic and maternal-zygotic mutants. Our results support the idea 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 at the optic chiasm.

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“…S2 " type="url"/> ; also note the presence of brain cells positive for fabp4a antisense probe without fli1:EGFP labeling). Neither of the other two probes generated similar patterns: no specific signal was observed with the fabp4a sense probe while the slit2 antisense probe labeled the ventro-medial part of the neural tube as reported previously ( Davison and Zolessi, 2021 ). These results corroborated the specificity of the fabp4a antisense probe and validated the post-WMISH immunofluorescence procedure and reagents.…”

“…To synthesize the probes we amplified the template using T7 and SP6 primers and afterwards generated digoxigenin (DIG) labeled probes by in vitro transcription with T7 or SP6 polymerases, using Digoxigenin-11-UTP (Merck). As an additional specificity control we used a slit2 antisense probe which has already been tested (generously provided by C. Davison, Facultad de Ciencias, Universidad de la República; Davison and Zolessi, 2021 ).…”

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“…S1; note the presence of brain cells positive for fabp4a antisense probe without fli1:EGFP labeling). Neither of the other two probes generated similar patterns: no specific signal was observed with the fabp4a sense probe while the slit2 antisense probe labeled the ventro-medial part of the neural tube as reported previously (Davison and Zolessi, 2020). These results corroborated the specificity of the fabp4a antisense probe and validated the post-WMISH immunofluorescence procedure and reagents.…”

“…To synthesize the probes we amplified the template using T7 and SP6 primers and afterwards generated digoxigenin (DIG) labeled probes by in vitro transcription with T7 or SP6 polymerases, using Digoxigenin-11-UTP (Merck). As an additional specificity control we used a slit2 antisense probe which has already been tested (generously provided by C. Davison (Davison and Zolessi, 2020).…”