Genes involved in eye development are highly conserved between vertebrates and Drosophila. Given the complex
genetic network controlling early eye development, identification of regulatory sequences controlling gene expression
will provide valuable insights toward understanding central events of early eye specification. The focus of this study is the
defining of regulatory elements critical for Drosophila eyes absent expression. Although eya has a complex
expression pattern during development, analysis of eye-specific mutations in the gene reveal a region selectively
deleted in the eye-specific alleles. Detailed analysis has been performed of a small 322 bp region immediately upstream of transcriptional start that is deleted in the eye-specific
eya2 allele. This analysis shows that this region can direct early eya gene expression in a pattern consistent with that
of normal eya in eye progenitor cells. Functional studies indicate that this element will restore appropriate eya
transcript expression to rescue the eye-specific allele. Regulation of this element during eye
specification has been examined, both in normal eye development and in ectopic eye formation. These studies demonstrate that the
element is activated upon ectopic expression of the eye specification genes eyeless and dachshund, but does not
respond to ectopic expression of eya or sine oculis. The differential regulation of this element by genes involved during
early retinal formation reveals new aspects of the genetic hierarchy of eye development (Bui, 2000).
The eya enhancer is expressed in ey, so, and dac mutant
eye discs in a pattern consistent with previous studies of
Eya protein expression during normal eye development. Normally, eya
expression is dependent upon ey activity, partially dependent
upon so activity, and independent of dac activity. Regulation during ectopic eye formation was addressed
in order to define genes that control the expression of this
eya enhancer region and to observe differential activation of
the eya enhancer. Activity of the enhancer was detected
upon ey- and dac-induced eye formation, as anticipated by
previous studies. However, enhancer activation is not
apparent upon ectopic eya or so gene expression or the
combination of eya and so together. Thus, this eya enhancer
appears to be selectively activated during ectopic
eye formation, indicating a molecular distinction in how ey
and dac genes induce ectopic retinal tissue compared to
induction by the eya and so genes, at least with respect to
regulation revealed by this element (Bui, 2000).
The regulation of this defined eye enhancer for eya
suggests that eya and so function distinctively, at least in part,
from dac and ey in ectopic eye formation. Whereas ey and
dac either directly activate or feedback to activate eya
expression, eya and so do not participate in regulatory loops
to the level of activation of eya gene expression as defined
by the eya eye enhancer (Bui, 2000).
eya can also synergize with dac in ectopic eye development,
and physically interact with the Dac protein. However, the loss-of-function phenotype of dac in the eye is not identical to that of eya and so. These studies also suggest that dac is not
acting the same way as eya with respect the eya enhancer: dac strongly activates expression,
but eya does not. Based on observations from expression studies, dac has previously been placed
downstream of eya. However, Dac is reduced, but not missing from eya mutant eye discs. The reduced expression may
reflect massive loss of eye progenitor cells in eya mutant
eye discs; alternatively, or in addition, there may be a partial
dependence of Dac expression upon eya gene function.
Thus, Dac may indeed be involved normally in aspects of
eya gene expression. Previous studies showing Eya expression
on ectopic eye formation are confounded by the fact
that Eya is expressed both prior to and after the appearance of the furrow, but
this expression is likely to be under the control of different
regulatory elements. The element defined here presents a
probe for at least some aspects of the early regulation of eya
gene expression. The functional requirement by eya for ey
and dac activity (and vice versa) in ectopic eye formation
may reflect concurrent roles or other, later roles of these
genes in eye formation. ey clearly has multiple roles at
distinct times in eye development, such as regulation of
genes important for late events of photoreceptor cell differentiation, in addition to the early function stressed here (Bui, 2000).
With respect to eya enhancer activation, ey and dac may
directly bind to the eya eye enhancer or the regulation may
be indirect through additional, yet-to-be defined genes. It is
suggested the regulation may not be direct, at least for Ey, as
Ey binding sites are not clearly apparent within the element. Whether Dac protein directly
binds to DNA has yet to be determined, but it likely interacts
with known transcriptional regulators in addition to interacting
with Eya. Yeast one-hybrid
experiments have also failed to support direct activation of
the eya enhancer by Dac or Ey (as well as confirmed lack of
activation by Eya and So).
These studies provide a framework from which to define
additional molecular genetic controls on early retinal specification.
Recent studies showing that the fundamentals of
ey/Pax-6 regulation can cross species boundaries suggests that not only are elements of the genetic
pathway controlling eye development conserved in vertebrates,
but fundamental aspects of the regulatory mechanisms
may also be conserved. Given that vertebrate Eya
homologs display functional rescue of Drosophila eya
mutants, key regulatory aspects of eya
gene expression, in addition to the function of the protein,
may also be conserved. Eya is a critical gene of eye formation,
with complex regulation of expression as shown here, as well as complex protein interactions, and multiple downstream targets. This eye enhancer controlling early
eya expression provides a molecular genetic tool to help
dissect additional regulatory events of eye specification that
are involved in the conserved pathways of eye formation (Bui, 2000).
According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial
cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited
ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three
developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions
where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker
genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus,
indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).
In contrast to ato broad expression just anterior to the
furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes
evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential
for uncommitted cells to acquire ommatidial cell fate or the
ability to express ommatidial marker genes. Consistent with
this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least
for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).
This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow.
Uncommitted cells just posterior to the morphogenetic
furrow are presumed to acquire ro expression potential at the earliest
stage of the model (stage 1). In stage 2, R3/R4 precursors expressing ro acquire svp
expression potential. svp expression in wild type
R3/R4 precursors along with Ras1 activation-dependent svp
misexpression in uncommitted cells is assumed to be not
only positively regulated by the concerted action of Ras1
signaling and Dac and Eya but also negatively regulated
by the protein product of the prepattern gene, lz.
R1/R6 photoreceptors are recruited into ommatidia between
stages 2 and 3. R1/R6 fate is previously shown specified by dual Bar homeobox genes, BarH1 and BarH2, whose expression is positively regulated by the cell-autonomous function of lz and svp. Consistent with this, in the putative R1/R6 arising area (around row 6), considerable svp expression occurs even in the presence of Lz. svp expression
is regulated by Dac and Eya, so that normal Bar expression
or R1/R6 fate eventually comes under the control of
putative eye prepattern genes Lz, Dac, and Eya (Hayashi, 2001).
In stage 3, which may correspond to R7 and cone cell
formation stages, pros is positively regulated through the
concerted action of Ras1 signaling and prepattern gene lz (Hayashi, 2001).
In the developing Drosophila eye, differentiation of undetermined
cells is triggered by Ras1 activation but their ultimate
fate is determined by individual developmental
potential. Presently available data suggest that developmental
potential is important in the neurogenesis of vertebrates
and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along
the dorso-ventral axis in response to graded Sonic Hedgehog
signals and this presages their future fates. Subdivision of originally
equivalent neural progenitors through the action of
prepattern genes may accordingly be a general strategy by
which diversified cell types are produced through neurogenesis (Hayashi, 2001).