The termination of retinal axons in the pretectal region of mammals

A systematic search for mutations affecting the retinotectal projection in zebrafish larvae was performed, as part of the large-scale Tubingen screen for homozygous diploid mutants in embryonic development. 2,746 inbred lines (F2 families) from males mutagenized with ethylnitroso urea were screened. In wild-type larvae, developing retinal axons travel along a stereotyped route to the contralateral optic tectum. Here, their terminals form a highly ordered retinotopic map. To detect deviations from this pattern, an axon tracing assay was developed that permits screening of large numbers of mutagenized fish. Two fluorescent tracer dyes (DiI and DiO) were injected at opposite poles of the eyes of day-5 aldehyde-fixed larvae. 12 hours later, retinal axons were labelled over their entire length, and could be observed through the intact skin. The assay procedure (aldehyde fixation, mounting, injection of dyes, microscopic analysis) took about 1 minute per fish. In total, 125,000 individual fish larvae were processed. During the screen, 114 mutations in approx. 35 genes were discovered. For the mutants subjected to complementation testing, the number of alleles per locus ranges from 1 to 15. The mutations affect distinct steps in the retinotectal pathway, from pathfinding between eye and tectum to map formation along the dorsal-ventral and the anterior-posterior axis of the tectum. Mutations that disturb axon pathfinding to the tectum for the most part do not disrupt retinotopic mapping, and vice versa. The majority of the mutants display associated defects in other tissues and die before day 10. These mutants provide new tools for studying the formation of neuronal maps. The results of this screen show that a large-scale genetic approach can be applied to relatively late and circumscribed developmental processes in the vertebrate brain.

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Zebrafish mutations affecting retinotectal axon pathfinding

Development ◽

10.1242/dev.123.1.427 ◽

1996 ◽

Vol 123 (1) ◽

pp. 427-438 ◽

Cited By ~ 25

Author(s):

R.O. Karlstrom ◽

T. Trowe ◽

S. Klostermann ◽

H. Baier ◽

M. Brand ◽

...

Keyword(s):

Ganglion Cells ◽

Fish Larvae ◽

Retinal Ganglion ◽

Axon Pathfinding ◽

Class Iii ◽

Axonal Projections ◽

Retinal Axons ◽

Complementation Groups ◽

Single Allele ◽

Neuronal Growth Cones

We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum. 82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline. The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all-or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.

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