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The Drosophila
compound eye represents an intriguing developmental paradigm
for understanding how instructions from signal transduction
cascades are superimposed upon vast regulatory networks of
transcription factors. The early development of the fly eye
can be divided into two broad phases: eye specification and
pattern formation. In a single eye disc preparation both events
can be observed simultaneously with specification occurring
anterior to the morphogenetic furrow and pattern formation
having taken place behind this mobile compartment boundary
(Figure 1). Ahead of the morphogenetic furrow cells are first
being asked to adopt a "retinal" fate as opposed
to a leg, wing, antennal or genital fate. Behind the furrow
these same cells are now then asked to group themselves into
organized units and to adopt individualized cells fates. Cells
on either side of the morphogenetic furrow will send and receive
instructions from the Ecdysteroid, RTK, Notch, Wingless, Hedgehog
and Decapentaplegic signaling pathways. These competing and
sometimes conflicting instructions are relayed to an impressive
array of specific DNA-binding proteins within the nucleus.
One of our interests is to determine how cells of the developing
eye sort through this apparent chaos of signaling and produce
a near perfect lattice of unit eyes that has been affectionately
referred to as a neurocrystalline array (Figure 1).
In a screen for
new genes that function during eye development we have identified
CREB Binding Protein (CBP) as playing a role in both the determination
of the eye itself and in the correct specification of individual
cell fates within the ommatidium. CBP, which is encoded by
the nejire (nej) locus, belongs to the CBP/p300
family of proteins. Over the past several years both Drosophila
and mammalian CBP have been shown to modulate transcription
and thus influence development by (1) remodeling chromatin
via acetylation and binding of histones and (2) serving as
a transcriptional co-activator by physically linking the basal
transcriptional machinery to a large array of specific DNA-binding
factors many of which are terminal members of signaling pathways.
Furthermore, CBP has the ability to interact with nuclear
hormone receptors and contains several zinc-finger binding
domains suggesting an active role in directing transcription
(Figure 2). Consistent with a role in development, human patients
with lesions within the CBP gene suffer from Rubenstein-Taybi
syndrome in which pattern formation proceeds incorrectly and
is characterized by severe facial abnormalities, broad thumbs,
broad big toes and mental retardation. Strabismus, cataracts,
juvenile glaucoma and coloboma of the eyelid, iris and lens
are among the eye defects associated with this syndrome. Similarly,
mutations within the Drosophila CBP homolog have
wide ranging pleiotropic phenotypes. A current focus of our
laboratory is to understand the role that CBP plays in the
specification and patterning of the fly eye.
We have used a
variety of approaches to demonstrate that CBP functions during
eye development. Immunohistochemical approaches have demonstrated
that CBP is present in all cells ahead and behind the morphogenetic
furrow. Phenotypic analysis of loss-of-function retinal mosaic
clones, viable heteroallelic combinations and expression of
RNAi constructs indicate that CBP is required for the expression
of several eye specification genes within the embryo and developing
eye imaginal disc while also functioning later during photoreceptor
cell fate determination (Figure 3). Furthermore, a series
of truncated CBP proteins, each lacking a unique set of protein
domains were expressed within the developing eye and appear
to function as "protein sinks" by soaking up transcription
factors that normally interact with CBP during retinal development.
This use of pathway interference has led to the identification
of roles for CBP within several photoreceptor cell subtypes
including the R7 neuron (Figure 3).
Although biochemical
approaches have identified nearly 100 proteins that physically
interact with CBP, we have pursued experiments that are aimed
at identifying proteins that interact with CBP during eye
development. Additionally, we are also interested in extending
these aims by identifying domains of CBP that mediate these
biochemical interactions. In order to accomplish these goals
we have expressed several truncated CBP proteins in developing
photoreceptor cells. The resulting rough eye phenotypes served
as starting materials for genetic screens that were designed
to isolate second site suppressor and enhancer mutations (Figure
4).
We
have been able to demonstrate genetic interactions between
CBP and a large number of proteins including members of the
Decapentaplegic and EGF Receptor pathways. Both cascades are
known to play significant roles in several phases of eye development
including retinal determination, morphogenetic furrow initiation
and cell fate specification. Interestingly we were also able
to identify a genetic interaction between CBP and the transcription
factor CREB-A. This interaction was identified by the ability
of mutations in CrebA to suppress the effects of overexpressing
CBP in the eye. Removal of CrebA from the developing eye leads
to the loss of photoreceptor cells while overexpression can
often lead to the initiation of ectopic eyes in tissue that
lies adjacent to the normal compound eye (Figure 5). |
