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1. Understanding the Function of the Human FOR/WWOX Tumour Suppressor Gene through Genetic Analysis of its Orthologue in Drosophila

This is a basic research project aimed at understanding the normal and cancer associated functions of the FOR/WWOX tumour suppressor gene identified by virtue of its chromosomal location (spanning fragile site FRA16D).

We have succeeded in mutating the Drosophila orthologue of the human FOR/WWOX gene and have commenced studies to understand the functional consequences of loss of FOR/WWOX activity. The Drosophila DmWWOX orthologue shares 49% amino acid identity with human WWOX and has a conserved domain structure of tandem WW domains and oxidoreductase homology. DmWWOX null mutants are viable and fertile with no obvious phenotype however, they do show an increased sensitivity to ionising radiation (O’Keefe et al., 2005). Protection from radiation can be restored by re-introduction of either Drosophila or human WWOX. This highlights the evolutionarily conserved function of WWOX and the utility of this model system for genetic dissection of the normal pathways of WWOX function. These results are also consistent with a protective role for WWOX in response to environmental challenges. The radiation sensitivity phenotype is similar to that previously observed for Drosophila mutants in p53 (11, 12). The in vivo significance of the reported interaction between WWOX and p53 can therefore be investigated using the Drosophila system. These studies will also allow the identification of additional pathway(s) in which DmWWOX normally participates and testing of the various reported in vitro interactions for their in vivo significance.

2a. Is there a Common Pathogenic Pathway in the Dynamic Mutation Diseases? – Studies in Drosophila

Several human genetic diseases are thought to be caused by polyglutamine repeats that expand in copy number beyond a threshold (e.g. Huntington’s disease). There are, however, numerous other neurodegenerative diseases that have very similar clinical symptoms to the polyglutamine diseases and are due to expanded repeats, but these repeats cannot encode polyglutamine. We have therefore used transgenic Drosophila models of these repeat expansion diseases in order to test hypotheses in regard to whether there might be common pathogenic pathways for dynamic mutation diseases.

We have specifically assessed 1) whether RNA might be the pathogenic agent [as has been shown for CUG expansion in myotonic dystrophy] 2) whether polyalanine might be the common agent (by virtue of frameshift slippage) 3) whether apoptosis initiated by DNA breakage of the expanded repeat (as is seen for rare chromosomal fragile sites) might be the common pathogenic pathway.

In each case the transgenic Drosophila analysis has been able to rule out the hypothesis being tested. Therefore the likelihood is that polyglutamine is the pathogenic agent for those diseases with the capacity to translate their expansion into polyglutamine and that a distinct pathogenic mechanism exists for those related diseases where the repeat is unable to encode polyglutamine. These results favour the view that the intrinsic properties of the polyglutamine containing proteins are likely to contribute to the pathology. Further experiments will be undertaken to validate this view.

2b. The Normal Role of Huntingtin in Development – Studies in Zebrafish (Danio rerio)

Repeat expansions in the HD gene (encoding the protein, huntingtin) are responsible for Huntington’s disease, however the normal function(s) and intrinsic properties of huntingtin are largely unknown. In transgenic mouse experiments, huntingtin has been shown to play role in early development as homozygous null mice exhibit defects in early development and display an apparent increase in apoptosis in the embryonic ectoderm, a layer that normally expresses huntingtin (Zeitlin et al., 1995). Zebrafish are an advantageous model in which to further investigate the normal function of huntingtin in early vertebrate development. They provide abundant numbers of optically transparent embryos that develop externally, making them easily accessible, and the orthologue, hd, has high homology to that of humans.

We have used morpholinos (synthetic antisense oligos) complementary to the 5’ untranslated region of ZHD mRNA, to ‘knockdown’ huntingtin protein synthesis by microinjecting embryos at the 1 to 4 cell stage. Either of two non-overlapping morpholinos give the same phenotype of diminished head and eyes, pericardial oedema, thin yolk extension, and disrupted pattern of pigmentation. We have also shown that mosaic expression of EGFP driven by the ZHD promoter region (containing the morpholino-specific sequences) can be blocked by either ZHD-specific morpholino, but not by a random control morpholino. This demonstrates that the ZHD-specific morpholinos bind the target region and block downstream protein expression. Because of the previously noted observations of increased apoptosis in huntingtin nullizygous mice, we have analysed the ZHD knockdown ‘morphants’ using the TUNEL assay to detect cells undergoing apoptosis. As in the null mouse, an increased level of apoptosis is observed in the morphants in comparison to embryos injected with control morpholino, suggesting a conserved anti-apoptotic function for huntingtin.

Careful analysis of the knockdown phenotype and the ZHDprEGFP transgenic fish will provide further insight into the function of huntingtin during early development and hopefully identify a pathway(s) in which the protein participates. This information may also help identify any factors that contribute to the tissue specificity of pathology observed in Huntington’s disease.

Postgraduate Research Projects Currently Available

The Mechanisms in Human Genetic Disease laboratory is interested in enquiries from any potential PhD applicants for projects in research areas described on these pages. For further details, please contact the lab head.

Mechanisms in Human Genetic Disease Laboratory

North Terrace Campus
Level 2, Molecular Life Sciences
The University of Adelaide
SA 5005


Robert Richards
T: +61 8 8313 7541
F: +61 8 8313 4362