| The
eye specification cascade in Drosophila is an excellent
experimental system for performing structure/function studies
on closely related proteins, in part, because several members
of the pathway have arisen from ancient duplication events.
For example the network contains the Pax6 genes eyeless
(ey) and twin of eyeless (toy),
the Pax6(5a) genes eyegone (eyg) and
twin of eyegone (toe) and the Six family members
sine oculis (so) and optix. The
Pax6 genes sit atop the core eye specification cascade while
the Pax6(5a) genes appear to function to promote cellular
proliferation during retinal development (Figure 1). Our interest
is in understanding the extent to which members of each gene
pair have retained or diverged functionally.
In
order to address this question we have created a series of
deletion and chimeric constructs that are being used to (1)
rescue loss-of-function retinal phenotypes and to (2) induce
retinal development in non-retinal tissues, a special feature
of several members of the eye specification cascade. In Drosophila,
loss-of-function mutations exist in only one (eyg)
of the two Pax6(5a) genes. Flies harboring an eye specific
eyg mutation have small eyes containing approximately
50 ommatidia. In an effort to determine the functional requirements
of individual domains of EYG protein we are attempting to
restore eye development to eyg mutants by introducing
wild type and deletion variants of EYG. We are extending these
studies by introducing wild type and deletion variant of TOE
as well as EYG/TOE chimeric proteins in an effort to determine
the degree to which these Pax6(5a) genes have been functionally
conserved. In addition, we are expressing each of the depicted
deletions and chimeric proteins within the developing antenna
in an effort to determine the domain requirements on ectopic
or extra eye development (Figure 2). The results from these
experiments are predicted to further our understanding of
how the Pax6(5a) genes influence eye development.
We
are also looking at the level of functional conservation/divergence
that exists between the Pax6 protein EY and TOY. We have taken
a similar approach to that of the Pax6(5a) genes in that we
have made a series of EY and TOY deletion variants and EY/TOY
chimeric proteins. Prior experiments from other laboratories
have indicated that EY is a more potent inducer of eye development
when expressed along the A/P axis of imaginal discs (via the
dpp-GAL4 driver). We wanted to understand the biological
basis that underpins this observation. To this end we have
expressed all deletion and chimeric proteins and have assayed
their ability to induce ectopic eye development in several
non-retinal tissues (Figure 3). We are using this assay to
guide our understanding of the evolutionary differences between
the two Pax6 proteins.
|
