The Role of atg18 in Signal Transduction Pathways During Drosophila Development
University of Delaware
8J16 and 9E6 are allelic mutations that disrupt Wnt/Wingless (Wg) or Hedgehog (Hh) signaling based upon their embryonic ‘lawn of denticles’ phenotype. Goals of this project were to identify the disrupted gene and determine its role in Drosophila developmental signaling. Complementation analysis revealed 8J16/9E6 are alleles of autophagy-specific gene 18, atg18, which plays a role in autophagosome-lysosome fusion. In yeast, atg18 negatively regulates endosomevacuole targeting, the yeast lysosomal equivalent. In Drosophila, endocytic machinery mutations that block lysosome targeting cause continuous signaling. Therefore, I hypothesized atg18 mutants should cause a buildup of lysosomes at the expense of endosomes and decrease signaling. To the contrary, Distalless (Dll), a long-range Wg target, was enhanced in 8J16/9E6 mutant tissue in Drosophila wing discs. Analysis of 8J16/9E6 germline clone (glc) mutant embryos confirmed atg188J16, atg189E6, and atg18KG03090 (atg18P) are allelic. Immunofluorescent staining of glc embryos showed the segmentation defect is due to loss of Wg expression at embryonic stage nine. 8J16/9E6 glc embryos also display severe CNS defects not strictly the result of Wg loss and thus, these mutation show pleiotropic effects. 8J16/9E6 are intronic point mutations not predicted to disrupt mRNA splicing. Additionally, mutants are temperature sensitive, yielding lethality at 25ºC, but are partially viable at 29ºC. qPCR revealed a decrease in atg18 transcript levels in 8J16/9E6 relative to wild type (WT). At the increased temperature, an average fivefold reduction was found in WT atg18 transcripts levels. Relative to WT, 8J16/9E6 show a slight increase of atg18 transcript levels at 29ºC, which may explain the rescue phenotype observed at this temperature in the mutants. In order to further address the effect of this temperature sensitivity on signaling in wing discs, clonal analysis was performed using a non-heat shock system, which may show evidence of a decrease in signaling in 8J16/9E6 mutant tissue. Further experiments are required to fully characterize these mutants and the mechanisms by which they are acting to cause these disrupted phenotypes.
atg18 , drosophila development , signal transduction pathways