Analysis of growth-defective mutations in Drosophila melanogaster

Abstract

Growth, an important process required for development in organisms, controls the cell size and number in a tightly controlled manner to produce organs and eventually the whole organism of appropriate size. The reverse of growth, termed autophagy, is the process of breakdown of intracellular proteins, lipids, organelles and cytoplasm. The functions of autophagy can be to provide a source of nutrients and energy during periods of starvation and it can also play a developmental role in the breakdown of tissues. Together, growth and autophagy are implicated in the insulin/TOR pathway and dFOXO. This thesis describes the analyses of two D. melanogaster genes, roquin (roq) and CG4753 (renamed agpat72Ea in this thesis), which sits cytologically adjacent to roq, to have implications in growth, and roq's implication in autophagy. The agpat72Ea gene is a putative member of the l-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family of acyltransferases. The role of AGPATs in producing phosphatidic acids (PA) has been implicated in regulating growth. Using imprecise P-element excision to generate mutants for roq, two alleles were generated: roqA2 whose deletions is confined to within the roq gene and roqY2, who other than deleting sequences in roq also has deletions spanning into the gene CG4753. When mitotic clone cells were generated, roqY2 exhibited cell autonomous growth defects while roqA2 did not, suggesting that mutations in agpat72Ea is the cause of the phenotype. Using recombineering to generate a genomic roq rescue construct and PhiC31 integrase mediated integration into the fly genome to rescue Roq in roqY2, an agpat72Ea specific mutant allele was created. Further analysis of the growth defects in agpat72Ea mutants revealed it was not the cause of the cell autonomous growth defects, suggesting a spontaneous mutation could be the cause of the growth defects observed in agpat72Ea mutants. Roq was first discovered in sanroque mice where a mutation in roq causes autoimmunity in these mice. Initial analysis of the Roq orthologue in D. melanogaster showed that roq mutants exhibited a slow growth defect that eventually led to late 3rd instar larval death, and initial overexpression of Roq in the wing imaginal discs resulted in overgrown wing discs. Mitotic roq mutant cells also exhibited a strong reduction in starvation-induced autophagy, suggesting a complicated role for Roq in regulating growth and autophagy. Studies described in this thesis have provided no evidence for a role for Roq in a growth pathway. However, this thesis has shown evidences for Roq's role in autophagy. The strong reduction in starvation-induced autophagy in mitotic roq mutant clones can be restored with the overexpression of dFOXO or S6 kinase (S6K), two molecules shown to be able to activate autophagy. This suggests a regulatory role for Roq in autophagy that is upstream of dFOXO and/or S6K function. Thus investigation of Roq in this thesis has revealed an important role for Roq in autophagy and possibly an evolutionarily conserved role in an early involvement in immunity.