Graham, Stephanie M. (2012) Using FlyAtlas to detect novel functions for well-known genes in Drosophila melanogaster. PhD thesis, University of Glasgow.Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.
Drosophila melanogaster has been an important model organism for over a century, cumulating in a vast array of mutant and transgenic stocks, the publication of the genome, its subsequent annotation and more recently the production of the online gene expression database, FlyAtlas. Much of what we know about developmental biology was pioneered in Drosophila and it is possibly the most well studied and understood model organism, in terms of development, genetics and physiology. The so-called ‘omics’ era of biology has resulted in a relatively data poor discipline quickly becoming a data rich one. Therefore the need for a good model organism, which offers the balance between genetic power and relevance has never been more important, as scientists begin to evaluate and analysis this data. We will argue that Drosophila melanogaster offers the best opportunity to study the relevance of omics data. FlyAtlas is an online resource, which allows scientists to look at tissue specific gene expression in the fruit fly Drosophila melanogaster. Unexpected expression patterns of previously characterised genes may hint at novel functions, thus helping to close the phenotype gap. To test this hypothesis we looked at the neuronal gene Fasciclin 2 (fas2), which has been exhaustively characterised (over 500 papers), with neural functions ranging from axonal growth in development to synapse stabilization in the adult. Surprisingly FlyAtlas showed fas2 is predominately expressed in the Malpighian tubule (a renal, rather than neural, tissue), hinting at a previously unreported function in this tissue. Results suggest fas2 may play an important role in apical microvilli development and stability in the principal cells of the tubules. We have also shown that Fas2 may be involved in actin localisation. Fas2 shows dynamic localisation in response to cAMP and over expression of the protein results in a significant increase in secretion when tubules are stimulated with cAMP. We also present evidence that Fas2 co-localises with F-Actin bundles in response to cAMP, hinting at a role for the actin cytoskeleton in secretion. Proteomics experiments carried out in order to determine Fas2’s, interacting partners proved problematic. For this reason 2D Blue Native PAGE and sucrose gradient techniques were optimised in order to facilitate this problem. 4 Unfortunately we were unable to isolate Fas2, however we have shown that BN-PAGE offers a robust protocol for the isolation of protein/protein complexes. We can also conclude from these experiments that 2D BN-PAGE offers an ideal comparative data source for transcriptomics data such as FlyAtlas. The second gene tested in this study is the sex determination transcription factor Doublesex (dsx). Dsx has been extensively studied in its role in differentiation of both the soma and to some extent the nervous system in males and females. FlyAtlas results indicate that it is also expressed in the Malpighian tubules, again hinting at previously unknown function in this tissue. Further to this the male and female transcripts of dsx are expressed in a sex specific manner. Our results confirm these observations and dsx was localised to the principal cells of the main and lower segments of the tubules. Male tubules however do not express dsx in the transitional segment whereas females do, suggesting that perhaps this segment of the tubule constitutes a previously unknown sex specific function. We have determined that Tra RNAi is effective at knocking down the female transcript in female tubules, allowing for the study of masculinised tubules in an otherwise female fly. Experiments concluded that although males and females show differential survival in response to bacterial infection, this is not controlled by dsx expression in the tubules. Preliminary results also suggest that two genes CG8719 and YP3 are differentially expressed in male and female tubules and offer ideal candidates to study dsx role in sexually dimorphic gene expression in the tubules. In conclusion this study verifies the use of FlyAtlas to determine novel functions for well-known genes in D.melanogaster. In turn this indicates the importance of omics data, as a staring point for further functional analysis of both genes and proteins.
|Item Type:||Thesis (PhD)|
|Keywords:||Drosophila melanogaster, malpighian tubules, FlyAtlas, Fasciclin 2, Doublesex, omics|
|Subjects:||Q Science > QP Physiology
Q Science > Q Science (General)
|Colleges/Schools:||College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology > Molecular Cell and Systems Biology|
|Supervisor's Name:||Dow, Prof. Julian A.T. and Davies, Prof. Shireen-Anne|
|Date of Award:||2012|
|Embargo Date:||13 April 2015|
|Depositing User:||Miss Stephanie Marie Graham|
|Copyright:||Copyright of this thesis is held by the author.|
|Date Deposited:||13 Apr 2012|
|Last Modified:||10 Dec 2012 14:05|
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