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Zusammenfassung:
Because pathways for axon guidance are sensed by growth cones through transmembrane proteins, we took an unbiased approach to reliably detect highly transient binding between cell-surface molecules. By taking advantage of the AVEXIS assay, we detected a novel interaction between the extracellular domains of membrane proteins Islr2 and the Vasorin paralogs. Paired expression analysis suggested a functional role for these molecules in retinotectal axon guidance: islr2 marks differentiated retinal ganglion cells, while Vasorin proteins are detected at the membrane along the complete retinal axon pathway. In particular, Vasnb marks a small set of midline glial cells at stages when the optic chiasm first forms. Contrary to rodents, zebrafish have completely crossed retinal axon projections. This makes the zebrafish an ideal model system to study default midline crossing decisions. We found that an islr2 TILLING mutant displays an ectopic ipsilateral projection, alongside variable thinning of the optic nerve. We generated a vasna targeted mutant by TALEN mutagenesis and analyzed a vasnb TILLING mutant, but preliminarily detected no phenotype in these larvae. After inducing deletions in the vasnb locus by the CRISPR/Cas9 system in the vasna mutant background, we are in the process of analyzing double mutants for the two paralogs. In addition, we are attempting to describe the islr2 guidance phenotype by live imaging of the retinal ganglion cell growth cones.
