Determining the Genotype-Phenotype Relationship of atg18a Mutants

Fischer, Matthew
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University of Delaware
Autophagy is a process where cytoplasmic components are engulfed by a phagophore, which then fuses with lysosomes to degrade its contents. This is a highly regulated and conserved process that is active during conditions of starvation and normal cellular maintenance. 8J16 and 9E6 are two independent allelic point mutations in Drosophila melanogaster autophagy-specific gene 18a (atg18a) located in introns three and four, respectively, of its five exon locus at 66B11 on chromosome 3. These atg18a alleles were initially characterized as being involved in Wnt or Hedgehog signaling due to their embryonic phenotype. The atg18a8J16 and atg18a9E6 intronic mutations are not in any sequences expected to disrupt transcriptional processing, yet these mutations cause lethality in homozygous germline clone embryos, pupal lethality in deletion heterozygotes, and neurodegenerative phenotypes. atg18a8J16 and atg18a9E6 mutants also show an unusual cold sensitive phenotype and are temperature permissive when reared at an elevated temperature. This is not observed in a third adult lethal allele, atg18aP, a P-element insertion of the construct KG03090 in the 2nd exon (5’ UTR) of atg18a. The exact molecular effects of these mutations are unknown. The purpose of this research is to investigate and determine the genotype-phenotype relationship of how atg18a8J16, atg18a9E6, and atg18aP alleles give rise to atg18a loss of function and characterize the effect of these mutations on neuronal maturation using the Drosophila eye as a model. Effects on splicing were examined by performing RT-PCR using primers that span introns of total RNA isolated from atg18P/ΔDF4408, atg188J16/ΔDF4408, atg189E6/ΔDF4408, and wild type xi i pupae. This analysis revealed no significant splicing defect, but a small effect was observed in atg189E6, which will be re-examined in the future. In adults, atg188J16 and atg189E6 homozygous mutant eyes are small and display the rough eye phenotype. To better understand the basis of this phenotype, mutant larval imaginal eye discs were examined to determine ommatidial organization and photoreceptor identity. ELAV, a pan-neuronal marker, showed that photoreceptors differentiate normally, but those that differentiate early are being lost. Examination of cleaved caspase 3 revealed that homozygous mutant photoreceptor cells were succumbing to apoptosis. It also showed significant death at the morphogenetic furrow and posterior compartment, which might result in a smaller pool of photoreceptor progenitors. Both of these phenotypes could contribute to the adult small, rough eye phenotype. These results suggest autophagy may be required for photoreceptor maintenance and its absence leads to apoptotic death. Examination of Senseless and Prospero expression showed that photoreceptors R8 and R7, respectively, are more refractory to apoptosis as these photoreceptors predominate near the optic stalk where the first photoreceptors differentiate. This suggests autophagy is more important for the maintenance of photoreceptors R1, R2, R3 R4, R5 and R6 than it is for R7 and R8.
Biological Sciences