Figure S2.

Effects of Rtnl1 mutant CRISPR alleles on Rtnl1 expression. (A) RT-PCR strategy to test presence of Rtnl1 transcripts (top). For the Rtnl1 isoforms shown, blue boxes indicate exons, broken lines indicate introns, light orange boxes indicate 5′ and-3′ UTRs, and arrows indicate the direction of transcription. A green triangle shows the position of the Rtnl1::YFP exon trap insertion (CPTI001291). Genomic coordinates are based on Release 6.26 of the D. melanogaster genome (http://flybase.org). Using the indicated primers, the expected amplicon from genomic DNA is around 14 kb. The expected amplicon for Rtnl1 transcript F is 636 bp, and for transcript H is 672 bp, due to an extra small exon (arrow). The box surrounded by a broken line shows the region containing Rtnl1¹⁸ lesions. Agarose gel electrophoresis (1%) of reverse-transcribed cDNA (bottom) shows that Rtnl1¹⁸ does not disrupt Rtnl1 mRNA transcription. For both WT and Rtnl1¹⁸ alleles, only one amplicon is seen, which is slightly smaller in Rtnl1¹⁸, due to the small deletions totaling 25 bp within the amplified region (see Fig. 1 A and Fig. S1 A for details). (B) Confocal sections of neuronal cell bodies, peripheral nerves and NMJs (muscle 1) show that Rtnl1::YFP³ mutation is enough to abolish Rtnl1::YFP expression. In cell bodies, nuclei are distinguished by the absence of α-HRP signal (dotted line regions). On the peripheral nerve, glia can be distinguished from neuronal axons due to the low levels of α-HRP signal (arrowhead). All larvae are also expressing Ib-GAL4, but this is not driving any reporter expression. Scale bars, 5 μm. Source data are available for this figure: SourceData FS2.