Analysis of the forkhead box subgroup 'O' (FOXO) family of transcription factors and the toxic effects of Ga14 in Drosophila melanogaster

Kramer, Jamie M. (2005) Analysis of the forkhead box subgroup 'O' (FOXO) family of transcription factors and the toxic effects of Ga14 in Drosophila melanogaster. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The insulin signalling pathway provides a conserved mechanism used by metazoan animals to regulate growth, metabolism, and behaviour in response to environmental cues. The FOXO transcription factors regulate cellular function in the presence of low levels of insulin signalling. This thesis describes the identification of the Drosophila melanogaster homologue of FOXO (dFOXO), that has high sequence conservation in the forkhead box DNA binding domain and Akt phosphorylation sites, when compared to analogous genes in mice, humans, and Caenorhabiditis elegans. Overexpression of dFOXO in the developing eye leads to a characteristic phenotype resulting from growth reduction. This phenotype can be rescued by co-expression of upstream PI3K signalling components, unless FOXO is mutated to be non-responsive to Akt phosphorylation. Ubiquitous expression of dFOXO at different stages of larval development closely replicates the effects of starvation, and endogenous dFOXO activity is modulated by the presence of nutrients. These results suggest that dFOXO is important for the response of Drosophila larvae to starvation. Analysis of dFOXO loss of function suggests that dFOXO is required for the adaptation of young larvae to starvation, but is not required during starvation in the later stages of larval development. A role for dFOXO in larval wandering is suggested by the observations that loss of dFOXO leads to reduced height of pupation, overexpression of dFOXO induces wandering, and endogenous dFOXO activity is increased during the wandering stage of the larval life cycle. In summary, dFOXO may regulate growth, behaviour, and starvation resistance in response to reduced PI3K signalling in Drosophila. -- Studies in Chapter 1.2 utilize the UAS/Gal4 ectopic expression system. We have shown that Gal4 causes a rough eye phenotype in adult flies when expressed at high levels during eye development. The rough eye phenotype is characterized by a loss of ommatidial organization and is accompanied by a high level of apoptosis in the eye imaginal discs where Gal4 is expressed. These phenotypes are essentially eliminated through co-expression of the caspase inhibitor, p35. This suggests that Gal4 has the capability to act as a toxic protein that activates the cellular apoptotic machinery. These results should be taken into consideration in the design and analysis of any experiment involving the use of the UAS/Gal4 system.

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