Zebrafish islr2 is expressed in RGCs and is necessary for complete retinal axon midline crossing. a Time-course analysis of islr2 mRNA expression in zebrafish embryos and larvae. At 36 hpf, the first time point at which the optic chiasm is established, islr2 is already present in RGCs (arrowheads). At 48 hpf the RGC layer is evenly populated by neurons expressing islr2. At 72 hpf we detected a slight decrease in the islr2 mRNA level and the presence of a subset of islr2-positive amacrine cells. Dashed outlines highlight retinas in ventral views. Upper row: ventral view. Lower row: lateral view. Anterior is to the left. b Schematic of the islr2 sa82 TILLING allele. The mutant ORF harbors a premature stop codon caused by a C-to-T mismatch, ultimately leading to a truncation of the LRR-CT domain. c . islr2 mutant larvae show ipsilateral retinal projections. First row: dorsal view of a wild-type larva at 5 dpf. The optic nerves from each eye (green and magenta) cross the midline to innervate the contralateral optic tectum (areas delimited by dashed lines). Second and third row: two homozygote mutant animals, displaying ectopic innervation of the ipsilateral tectum (asterisks). The larva in row 2 additionally shows a thinner optic nerve (arrow), while the individual in row 3 has optic nerves with normal width. All pictures are dorsal views and maximum intensity projections of confocal Z-stacks. Anterior is to the left. Scale bar: 50 μm. d Direct relationship between optic nerve width (brackets) and ipsilateral misrouting of retinal axons (arrowheads) at the chiasm of islr2 mutant larvae. Homozygous mutant individuals with normal optic nerve (second column) display a higher number of ipsilateral fibers, compared to mutants with thinner nerves. Single optical sections. Ventral views. Anterior is to the top. ON: optic nerve. Scale bar: 50 μm. d' Phenotype distribution in islr2 mutants compared to siblings. Joint analysis of two clutches of animals showing mostly unaltered optic nerve width. Mutant larvae (black bars) display a higher number of ectopic neurites in the ipsilateral optic tectum, compared to controls (grey bars) and to larvae with thinner optic nerves (compare to d''). The innervation area corresponding to the contralateral optic tectum was unaffected in mutants compared to siblings. CL: contralateral. d'' Joint analysis of two clutches where larvae with thinner nerves were identified. Only a subset of mutant animals (black bars) displays ipsilateral innervation of the optic tectum, while in many mutants these neurites are undetectable, likely because of the extreme reduction in retinal fiber number. In most mutant animals the innervation of the contralateral optic tectum is reduced, suggesting that thinning of the optic nerve and ipsilateral projections are independent phenotypes. CL: contralateral
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