Supplementary Figure 6: Characterization of SelR loss-of-function mutants and additional analyses of SelR axon guidance defects.
From: SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics
a, Loss-of-function SelR mutations. Several transposable element mutations are situated within SelR. In addition, transposable element mutations that contained FRT sites, e00293 and d04974, were identified as situated in genes that flanked the SelR locus. Using a FLP-FRT strategy20, we employed these two transposable element alleles to delete the region between d04974 and e00293 and generate the SelRDelta3 allele. Crosses between SelRDelta3 and DfExel6159 and DfExel7305 are semi-lethal. Crosses between SelRDelta3 and DfExel7306 (which does not remove SelR; see diagram) exhibit mendelian ratios. b-d, Further presentation of axon guidance defects found in SelR neuronal overexpression and SelR−/− mutants. For reference, the nerves projecting into each hemisegment are numbered (1, 2, 3) as are the intersegmental nerve (ISN) axons. b, Further characterization of neuronal overexpression of SelR and the guidance of model axons including those within the Drosophila intersegmental nerve b (ISNb) and segmental nerve a (SNa). In wild-type embryos, ISNb axons correctly innervate their muscle targets (open arrows). In contrast, neuronal overexpression of SelR (Neuronal SelR) generates highly penetrant axon guidance defects in which ISNb axons often fail to innervate their muscle targets (closed arrows). We also observe similar highly penetrant SNa axon guidance defects in which 69% of the hemisegments were affected and 81% of these defects resemble Semaphorin−1a−/− 21, PlexinA−/− 22, and Mical−/−1,19,23 mutants. Replicated in at least 2 independent experiments (separate crosses) per genotype. Scale bar in b applies to both images. Genotype of the SelR neuronal overexpression embryos was: Elav-GAL4/+; UAS: SelR/+. c, Further characterization of SelR−/− mutant axon guidance defects. c1, Multiple different axon guidance defects are seen in SelR−/− mutants including a lack of ISNb innervation of muscles 6/7 and 12/13 (arrows), abnormal fasciculation/clumping of ISNb axons (arrowhead), and a paucity of fasciculated/bundled ISNb axons (axons in hemisegment 2). c2, SelR−/− mutants also exhibit CNS axon guidance defects including abnormal midline crossing (open arrowheads). SelR−/− = SelRDelta3/DfExel7305 for these images. d, Increasing the levels of Mical in neurons generates motor (closed arrowheads) and CNS (open arrows and open arrowheads) axon guidance defects (Neuronal Mical image and graph; 1). The CNS from Figure S6d (Neuronal Mical) is a portion of the same CNS as that presented in Figure 6c. Expressing SelR in neurons in combination with Mical significantly rescues these axon guidance defects (Neuronal Mical + Neuronal SelR image [open arrows] and graph). Chi-Square Test; ***P<0.0001. n = 80 hemisegments assessed in 8 animals per genotype. Replicated in at least 2 independent experiments (separate crosses) per genotype. Scale bar in d applies to both images.
