Baier H, Klostermann S, Trowe T, Karlstrom R O, Nüsslein-Volhard C, Bonhoeffer F
Abteilung Physikalische Biologie, Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany.
Development. 1996 Dec;123:415-25. doi: 10.1242/dev.123.1.415.
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.
作为胚胎发育中纯合二倍体突变体大规模图宾根筛选的一部分，我们对影响斑马鱼幼虫视网膜 - 脑顶盖投射的突变进行了系统搜索。我们筛选了2746个用乙基亚硝基脲诱变的雄性自交系（F2家族）。在野生型幼虫中，发育中的视网膜轴突沿着固定的路径到达对侧视顶盖。在这里，它们的末端形成高度有序的视网膜拓扑图。为了检测与这种模式的偏差，我们开发了一种轴突追踪试验，可用于筛选大量诱变鱼。将两种荧光示踪染料（DiI和DiO）注射到第5天醛固定幼虫眼睛的相对两极。12小时后，视网膜轴突在其全长上被标记，并且可以通过完整的皮肤观察到。该试验程序（醛固定、固定装片、染料注射、显微镜分析）每条鱼大约需要1分钟。总共处理了125000条个体鱼幼虫。在筛选过程中，发现了大约35个基因中的114个突变。对于进行互补测试的突变体，每个基因座的等位基因数量范围为1至15。这些突变影响视网膜 - 脑顶盖通路中的不同步骤，从眼睛和脑顶盖之间的路径寻找，到沿着脑顶盖背 - 腹轴和前 - 后轴的图谱形成。大多数干扰轴突向脑顶盖路径寻找的突变不会破坏视网膜拓扑映射，反之亦然。大多数突变体在其他组织中表现出相关缺陷，并在第10天之前死亡。这些突变体为研究神经元图谱的形成提供了新工具。该筛选结果表明，大规模遗传方法可应用于脊椎动物大脑中相对较晚且有限的发育过程。
