Crumbs alternative splicing is disrupted in obelus mutants. (A) Crumbs-PA contains 28 EGF-like repeats, three lamin AG–like domains, and a transmembrane domain. Crumbs-PC contains an additional EGF-like repeat after repeat 7. (B) Schematics of crumbs splice variants expressed in obe mutants, numbered according to Flybase (dos Santos et al., 2015). Orange, alternative exon included in crumbs-RC. Gray, 5′ and 3′ UTRs. Introns are not to scale. (C) RNA sequencing read density in RPKM (reads per kilobase per million mapped reads) across exon 4 of crumbs and its flanking exons in wild-type (WT; gray), obe¹/obe² (dark blue), and obe²/Df (light blue) embryos at late stage 5/early stage 6. (D) Total crumbs mRNA levels in wild type (13,352 ± 1,061) were not significantly affected in obe¹/obe² (10,800 ± 881.4; P = 0.13) and obe²/Df (15,905 ± 488.3; P = 0.09). n.s., not significant. (E) The percentage of crumbs-RA in wild type (81 ± 4%) is significantly reduced in obe¹/obe² (35 ± 4%; P = 0.0013 by unpaired t test) and obe²/Df (18 ± 3%; P = 0.0002 by unpaired t test; three biological replicates per condition). Means ± SD (C) or ± SEM (D and E) between biological replicates are shown. The obe¹/obe² and obe²/Df mutant embryos were the progeny of obe¹/obe² and obe²/Df(3R)Exel6174 females, respectively, crossed to obe²/+ males. See also Table S1 and Fig. S4.