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Determining the role of a candidate peptide hormone ligand-receptor pair during pharate adult development in Drosophila
Publication . Varela, Ednilson Mascarenhas; Gontijo, Alisson; Homem, Catarina; Herédia, Fabiana
Insulin-like peptides (Ilps) of the Relaxin family have important functions in reproduction, development, and nervous system function across species and signal via Relaxin-specific G protein coupled receptors (GPCRs). In Drosophila, Ilp7 is the most highly conserved amongst the eight described Ilps, but its biological function remains unknown. The Drosophila Leucine-Rich Repeat-containing GPCR 4 (Lgr4) is a clear ortholog of the vertebrate Relaxin receptors, but which Drosophlia Ilp, if any, acts as its ligand, remains to be determined. Based on our evolutionary phylogenetic analysis I hypothesize that Ilp7 and Lgr4 form a ligand-receptor pair in Drosophila. Here, we incorporated GAL4 and LexA reporter genes in the endogenous Lgr4 locus (Lgr4::P2A::GAL4 or Lgr4::P2A::LexA, respectively) and used these reporters to infer Lgr4 expression patterns during Drosophila development. We find that most of the Lgr4-positive (Lgr4+) cells in the central nervous system (CNS) express the RNA-binding protein elav´ (embryonic lethal, abnormal vision), a postmitotic neuron marker, suggesting that most or all Lgr4+ cells are neurons, not glial or other cells in the CNS. By analyzing sequence data of our GAL4 and LexA alleles, we found out that there are two previously unnoticed Lgr4 alleles segregating in Drosophila stocks. Differences in Lgr4 expression patterns are observed between these alleles, particularly in the optic lobe. Neuroanatomical characterization identifies notable Lgr4+ cells in the pars intercerebralis (PI), distinct from insulin-producing cells (IPCs), suggesting functional significance. Lgr4 expression is also detected in the proventricular ganglion (PVG), ABLKs, and the gustatory systems, indicating extensive expression in both central and peripheral nervous systems with potential diverse functions. We show that mutants in either Ilp7 or Lgr4 do not exhibit obvious defects under normal laboratory conditions regarding development, fertility, and fecundity. However, both Ilp7 and Lgr4 mutants display aberrant light-avoidance behavior and play a crucial role at the end of late third-instar larvae, driving them to select a dark environment to pupariate. Moreover, we biochemically validated that Ilp7 and Lgr4 co-immunoprecipitate when epitope-tagged-versions of both proteins are ectopically co-expressed in cultured Drosophila S2 cells in vitro. Furthermore, we confirm that confinement induces a delay in D. melanogaster wing expansion using a different setup (our small chambers in the filming arenas), and we found that flies of the Ilp7 mutant stock (Ilp7[1]) fail to perform wing expansion behavior under confinement. Nevertheless, placing the Ilp7[1] mutation in another genetic background (RAL486) eliminated this effect.
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Fundação para a Ciência e a Tecnologia
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Concurso de avaliação no âmbito do Programa Plurianual de Financiamento de Unidades de I&D (2017/2018) - Financiamento Programático
Número da atribuição
UIDP/00329/2020