Genetic Mapping and Phenotypic Analysis of GstE14 E.4.1 on Eye and Antennae Development in Drosophila melanogaster

Abstract

Genetic screens are valuable for identifying novel genes involved in the regulation of developmental processes. To identify genes associated with cell growth regulation in Drosophila melanogaster , a mutagenesis screen was performed. Undergraduate students participating in Fly-CURE phenotypically characterized the E.4.1 mutant which is associated with rough eyes and antennae overgrowth. Following complementation analysis and subsequent genomic sequencing, E.4.1 was identified as a novel mutant allele of GstE14 , a gene involved in ecdysone biosynthesis important for the timing of developmental events. The abnormal eye and antenna phenotypes observed resulting from the loss of GstE14 suggest its role in tissue growth.

Figure 1. Characterization of the lethal GstE14 ^E.4.1 mutation by phenotypic analysis, complementation mapping, and genetic sequencing

(A) FRT42D, Dark ⁸² control mosaic eye (B) and FRT42D, Dark ⁸² , GstE14 ^E.4.1 mutant mosaic eye showing white (wildtype, white arrow) and red (mutant, black arrow) pigmentation as a result of FRT/FLP mitotic recombination during development. (B) Overgrowth of mutant tissue in genotype FRT42D, Dark ⁸² , GstE14 ^E.4.1 is observed as clusters of red pigmentation, in addition to mutant clones displaying necrotic tissue (black arrowhead) in mosaic eye and rough eye phenotype with disorganized ommatidial arrangement (white arrowhead). (C) FRT42D, Dark ⁸² control fly shows wildtype antennae. (D) FRT42D, Dark ⁸² , GstE14 ^E.4.1 mutants exhibit antenna overgrowth (arrow). (E) The narrowest region in which E.4.1 failed to complement in the genomic region 2R:13,219,130..13,249,241 (Image adapted from JBrowse on FlyBase). (F-G) Sanger sequence analysis of wildtype GstE14 and mutant GstE14 ^E.4.1 reveals a heterozygous peak of C → T at 2R:13,240,692. (H) Alignment of amino acids of control FRT42D, Dark ⁸² and mutant FRT42D, Dark ⁸² , GstE14 ^E.4.1 sequence show the presence of a nonsense mutation at amino acid 210 (Gln → Stop) in the GstE14 ^E.4.1 mutant resulting in a truncated protein missing the last twenty two amino acids in the C-terminal region of GstE14.