| The
Six family of DNA binding proteins plays a crucial role during
eye and head development in diverse animals that range from
fruit flies to mammals. Each member of this family contains
a homeodomain for DNA binding and a SIX domain for protein-protein
interactions. The genome of Drosophila contains three members
of this family: sine oculis (so), optix,
and D Six4 (Figure 1). These elements are thought
to have arisen through two independent duplications of a single
ancestral gene. D Six4 is involved in gonad and muscular
development, thus is outside the scope of our lab's work.
My research focuses on sine oculis and optix,
both of which appear to be part of a complex regulatory network
involved in the specification of the compound eye (Figure
2).
However,
the existence of many intriguing differences between these
two Six family members has prompted the close examination
of their respective roles in eye development. First, while
sine oculis is expressed throughout the entire developing
eye disc, optix expression is restricted to regions
anterior of the advancing morphogenetic furrow (Figure 3).
Second, expression of optix but not sine oculis
is sufficient to direct eye formation in non-retinal tissues
such as the developing legs. Finally, both genetic and biochemical
experiments have suggested that sine oculis interacts
with eyes absent while no such interaction has been
observed for optix.
I
would like to gain a better of understanding of how the sine
oculis and optix genes have evolved since their
birth. To this end I have created sets of reagents that will
test the roles that both genes play in eye formation. First,
I created a set of SO-OPTIX protein chimeras in which individual
domains of SO have been replaced with the corresponding regions
of OPTIX (Figure 4). The ability of each chimeric protein
to rescue the no-eye phenotype of the sine oculis null
mutant has been assayed (Figure 5).
Second,
I have created a set of SO mutant proteins in which the individual
domains of the molecule have been deleted (Figure 6). These
deletion proteins have also been assayed for their ability
to rescue the sine oculis loss-of-function mutant
phenotype. Together these chimeras and deletion proteins have
shed some light on the requirements for SO activity and on
the evolutionary conservation between sine oculis
and optix.
I
have found that the N-terminal Domain of SO is not necessary
for proper protein function, as the N-terminal Chimeric protein
and the SO N-terminal Deletion molecule were both able to
rescue the sine oculis mutant phenotype to near wild
type levels. The C-terminal Domain of SO also appears to be
dispensable; the C-terminal Deletion molecule gave strong
rescue of the no-eye phenotype as well. Interestingly, the
Homeodomain Chimeric protein was able to partially rescue
the sine oculis mutant. This leads me to believe
that the homeodomains of both SO and OPTIX may be binding
to similar DNA targets (Figure 7).
The
SIX Domain of SO is both necessary for proper protein function
and unique to the transcription factor. Neither the SIX Domain
Chimeric protein nor the SIX Deletion protein were able to
rescue the sine oculis mutant. Deleting this domain
abolishes critical interactions with partner proteins, thus
inactivating the molecule. Substitution of the domain renders
SO unable to recruit the proper protein cofactors.
During
the course of this experiment, I observed that three separate
constructs repressed eye development when over-expressed behind
the morphogenetic furrow using the GMR-GAL4 driver. Full length
optix, the so/optix C-terminal Chimera,
and the so/optix N- and C-terminal Chimera all caused
a small, severe rough eye when misexpressed (Figure 8). The
only domain that these three molecules share in common is
the C-terminal Domain of optix.
I
have created a set of constructs and am currently carrying
out experiments to answer several questions: 1) which domains
of optix, other than the C-terminal Domain, are required for
inhibition? 2) are specific regions of the C-terminal Domain
responsible for the observed inhibition? 3) if specific regions
can be identified, have they been conserved in the vertebrate
homologues SIX 3 and SIX 6? The answers to these questions
with begin to tease apart an important functional difference
optix and its cousin sine oculis.
|
