The Six genes encode a group of transcription factors that are characterised by a Six
domain believed to be required for protein interaction, and a homeodomain necessary
for DNA binding. Bioinformatics analysis, carried out in this PhD, shows the
evolutionary sequence conservation of these genes from poriferans to mammals.
There are three Six genes in Drosophila and six Six genes in mammals. Six genes in
mammals are involved in myogenesis and neurogenesis. SIX1, SIX4 and SIX5 are
coexpressed in mouse myogenesis and SIX1 and SIX4 are expressed in sensory
neurons early in trigeminal gangliogenesis. SIX3 is expressed in the developing eye
and the forebrain and ectopic expression of SIX3 results in the formation of the
retina. Six genes are also involved in human diseases. SIX1 is involved in the
branchio-oto-renal syndrome, SIX3 in holoproencephaly and the disruption in
expression of the SIX5 gene is thought to be involved in myotonic dystrophy.
The Drosophila melanogaster homologue of SIX5, D-Six4, when mutated
results in embryonic defects both in muscle and gonad development which are
thought to mirror the symptoms of myotonic dystrophy patients which include the
inability to relax muscles after contraction, cataracts, mental deficiencies and
sterility. D-Six4 is part of a family of three Six genes (D-Six4, sine oculis, Optix).
Mutations in these genes result in different phenotypes. Sine oculis and Optix are
expressed in the head and the developing eye. They are required for Drosophila eye
development. D-Six4 is expressed in the mesoderm, it is required for muscle and
gonad developments. Sequence identity, observed in the Six domain and
homeodomain of Six gene orthologues, has led to the grouping of Six proteins into
three subfamilies, Sixl/2, Six4/5 and Six3/6.
The aims of this PhD were to determine whether Drosophila is a good model for
studying Six genes and to investigate the conservation and divergence of function of
the different Six genes in Drosophila melanogaster. Firstly, I carried out sequence
comparisons of different Six proteins from poriferans to mammals and identified
features of conservation with putative functional importance. Importantly, I proposed
specific criteria defining the Six domain more accurately. I then hypothesised that the
different SIX proteins are functionally distinct and that these functional differences
are conserved between species. In order to test this, the ability of one Six protein to
substitute for the loss of function of another Six protein was assessed through genetic
rescue experiments. I found that Optix and Sine oculis can substitute for the loss of
function of D-Six4 in the muscle but not very efficiently and Sine oculis can partially
compensate for loss of D-Six4 in the gonad. This also gave insight into the role of DSix4
in mesoderm development. Finally, I sought to investigate the ability for a
vertebrate orthologue to substitute for a mutation of a Drosophila Six gene but due to
experimental difficulties that could not have been anticipated this was not achieved.
While the ability of only the three Six genes of Drosophila to complement the muscle
and gonad phenotypes in a D-Six4 mutant was assessed, molecular and fly work
carried out using mammal Sixl, Six4, Six5 and Six3 DNA resulted in invaluable
learning outcomes with regards to my training as a scientist.
The first three results chapters, chapter 2, 3 and 4, are bioinformatics analyses
of the protein sequence comparisons carried out throughout the metazoan phylum.
The ability for the Drosophila melanogaster paralogues of D-Six4 (Sine oculis and
Optix) to rescue aspects of the D-Six4 mutant phenotype are then discussed in
chapter 5. Chapter 6 discusses the work that was carried out in attempting to generate
molecular constructs with the vertebrate orthologues eg. Sixl, Six4 and Six5 genes.