GENETIC DISSECTION OF THE RETINOTECTAL PROJECTION

A systematic search for mutations affecting the retinotectal projectio n in zebrafish larvae was performed, as part of the large-scale Tubing en screen for homozygous diploid mutants in embryonic development, 2,7 46 inbred lines (F-2 families) from males mutagenized with ethylnitros o urea were screened, In wild-type larvae, developing retinal axons tr avel along a stereotyped route to the contralateral optic tectum, Here , their terminals form a highly ordered retinotopic map, To detect dev iations from this pattern, an axon tracing assay was developed that pe rmits 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 lab elled over their entire length, and could be observed through the inta ct skin, The assay procedure (aldehyde fixation, mounting, injection o f 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 subject ed 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 alon g the dorsal-ventral and the anterior-posterior axis of the tectum, Mu tations that disturb axon pathfinding to the tectum for the most part do not disrupt retinotopic mapping, and vice versa, The majority of th e mutants display associated defects in other tissues and die before d ay 10, These mutants provide new tools for studying the formation of n euronal maps, The results of this screen show that a large-scale genet ic approach can be applied to relatively late and circumscribed develo pmental processes in the vertebrate brain.