| dc.description.abstract | The expression of the Drosophila melanogaster morphogen Hedgehog (Hh) plays a
key role in co-ordinating proliferation and differentiation during animal
development. Tight spatial and temporal regulation of Hh expression is essential for
its correct function in these essential processes. Both mis-expression of its
mammalian orthologue Sonic Hedgehog (Shh) and aberrant stimulation of the
associated signalling pathway occur in a wide range of human tumours. Although
there is extensive knowledge of the signal transduction pathway that is activated in a
Hh-stimulated cell, very little is known about pathways governing the expression of
the morphogen itself.
The Drosophila tumour suppressor protein Hyperplastic Discs (Hyd), an E3 ubiquitin
ligase, negatively regulates hedgehog (hh) expression and Hh pathway activity by
independent mechanisms in the developing Drosophila eye. Genetically generated
hyd mutant clones in the eye mis-express hh and the transcriptional activator of Hh
target genes, Cubitus interruptus (Ci), and cause overgrowth of the surrounding wildtype
tissue. However, the underlying molecular mechanism(s) by which Hyd
regulates these morphogen regulatory pathways is not known. Hyd may be involved
in ubiquitylating target proteins in these pathways, which could have degradative or
non-degradative outcomes. In order to elucidate Hyd’s molecular role in potential
morphogen regulatory pathways, I applied a proteomics-based approach to identify
novel Hyd binding partners and ubiquitylated substrates. Tandem affinity
purification in combination with mass spectrometry was used to purify and identify
Hyd and its complexed binding partners from Drosophila cells. Binding and
ubiquitylation assays were subsequently used to verify and characterize the
interactions. In addition, a biased, literature-guided approach was applied to identify
likely Hyd binding partners based on their involvement in morphogen signalling and
conservation across species. Finally, to assess the functional consequences of a
newly identified interaction, I used a Drosophila in vivo model to determine whether
the novel binding partner was capable of modifying the hyd mutant phenotype. For
this purpose, the Mosaic Analysis with a Repressible Cell Marker (MARCM)
technique was used to generate hyd mutant clones in the developing larval eye,
which were expressing transgenes resulting in either the over-expression or RNAi-mediated
knockdown of the gene of interest.
My results indicate that Hyd is involved in regulating both Hh and Wg morphogen
signalling in the Drosophila eye, and that the molecular mechanism of action may, at
least in part, involve the protein kinase Shaggy (Sgg). Hyd interacts with the Hh and
Wg transcriptional activator proteins Ci and Armadillo, respectively, as well as the
Sgg kinase. Sgg is a negative regulator of both the Hh and Wg pathways, and acts to
direct the proteolytic processing or degradation of the transcriptional effectors of
these morphogen pathways. Sgg and its mammalian orthologue GSK3β were
ubiquitylated in vitro, and GSK3β ubiquitylation was negatively regulated by the
mammalian homologue of Hyd, EDD. Knockdown of sgg in eye disc cells mutant for
hyd resulted in a dramatic rescue of the overgrowth phenotype. Loss of hyd in clones
located in the anterior region of the eye disc resulted in low levels of the full-length
Hh transcriptional activator protein Ci. This effect was reversed completely as a
result of sgg knockdown. Furthermore, loss of hyd in eye disc clones resulted in
elevated Hh and Wg morphogen expression. Mis-expression of hh in hyd mutant
clones was significantly reduced upon over-expression of a constitutively active Sgg
kinase. Hence sgg appears to genetically act downstream of hyd to regulate hh gene
expression and Ci expression.
In summary my results identify Sgg as a novel regulator of hh gene expression,
whose activity may be regulated by ubiquitylation, and which may be acting
downstream of Hyd in a ubiquitin-regulated manner to control both hh gene
expression and Hh pathway activity in the developing Drosophila eye. Hyd may also
regulate Hh pathway activity by directly interacting with Ci and affecting its activity.
The results also indicate that Hyd may be a master regulator of both Hh and Wg
morphogen signalling during Drosophila development. | en |
