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).
Direct control of the proneural gene atonal by retinal determination factors during Drosophila eye development
The determination of neuronal identity in Drosophila cells depends on the accurate expression of proneural genes. The proneural gene atonal (ato) encodes a basic-HLH protein required for photoreceptor and chordotonal organ formation. The initial expression of ato in imaginal discs is regulated by sequences that lie 3' to its open reading frame. This report shows that the initial ato transcription in different imaginal discs is regulated by distinct 3' cis-regulatory sequences. The eye-specific ato 3' cis-regulatory sequence consists of two distinct elements termed 2.8PB and 3.6BP that regulate ato transcription during different stages of eye development. The 2.8PB enhancer contains a highly conserved consensus binding site for the retinal determination (RD) factor Sine oculis (So). Mutation of this So binding site abolishes 2.8PB enhancer activity. Furthermore the RD factors So and Eyes absent (Eya) are required for 2.8PB enhancer activity and can induce ectopic 2.8PB reporter expression. In contrast, ectopic Dpp signaling is not sufficient to induce ato 3' enhancer activation but can induce increased levels of RD factor Dachshund (Dac) and synergize with So and Eya to increase ato 3' enhancer activity. These results demonstrate a direct mechanism by which the RD factors regulate ato expression and suggest an important role of Dpp in the activation of ato 3' enhancer is to regulate the levels of RD factors (Tanaka-Matakatsu, 2008).
In addition to RD factors, Dpp signaling is also known to be involved in eye development although little is known about its role in the activation of the ato 3′ enhancer. This study found that induction of the ato 3′ enhancer by ectopic expression of So and Eya under the 30A-GAL4 driver was limited mainly to specific regions near the A/P compartment boundary where endogenous Dpp is expressed. In addition, co-expression of Dpp with So and Eya led to expansion of ectopic ato 3′ reporter expression, indicating that Dpp signaling can synergize with So and Eya to activate the 2.8PB enhancer. As the 2.8PB enhancer does not contain Mad binding sites, it is unlikely that Dpp signaling regulates 2.8PB expression directly through binding of Mad protein to 2.8PB. It is hypothesized that some of the downstream targets of Dpp signaling may mediate the ability of Dpp signaling to synergize with So and Eya in the activation of the ato 3′ eye enhancer. Interestingly, Dac, a RD factor regulated by Dpp signaling, can also synergize with So and Eya in activating the ato 3′ eye enhancer, raising the possibility that induction of Dac contributes to the ability of dpp to synergize with so and eya in the activation of ato 3′ enhancer. The level of Dac in the posterior of the wing disc is significantly lower than that in the anterior in the absence of Dpp co-expression, while similar levels of Dac in the anterior and the posterior are observed when Dpp is co-expressed. Therefore the difference in the subset of cells induced to activate the ato 3′ enhancer by dpp + so + eya and by dac7c4 + so + eya expression could be in part due to differences in the level of Dac induced by Dpp expression and that reached with the 30A-GAL4 driver. Alternatively, it is possible that Dpp signaling has additional targets that contribute to its synergistic induction of the ato 3′ enhancer with So and Eya (Tanaka-Matakatsu, 2008).
During Drosophila sensory organ formation, transcriptional regulation of the proneural gene ato plays a key role to determine the position of proneural clusters. Tissue-specific expression of ato is governed by the flanking cis-regulatory regions immediately upstream (5′) and downstream (3′) of the ato transcription unit. ato 5′ transcription largely depends on the Ato-dependent autoregulatory mechanism, while the ato 3′ cis-regulatory region appears to encode tissue- and temporal-specific information. This analysis of the ato 3′ cis-regulatory region revealed a modular organization of tissue-specific enhancers, each of which determine the initial ato expression in sensory organ precursors of a specific tissue type for the formation of ch organs or photoreceptors. For example, the 1.7 kb BamHI–StuI fragment immediately downstream of the ato transcription unit controls ato expression specifically in the leg discs while the 1.9 kb StuI–PstI fragment located 1.7 kb downstream of the ato transcription unit regulates ato expression specifically in the antennal ch organ precursors. Similarly, the eye enhancer lies within the BglII–PstI–EcoRI fragment located 2.8 kb downstream of the ato transcription unit. Finally the 1.5 kb EcoRI–BamHI fragment located 4.8 kb downstream of the ato transcription unit regulates ato expression during embryonic development (Tanaka-Matakatsu, 2008).
Taken together, these results demonstrate that the modular organization of the ato 3′ cis-regulatory region determines the spatial control of ato expression in the ch organs and photoreceptors in different imaginal discs. A surgical experiment of eye disc fragments has revealed that cells immediately anterior to the MF have already acquired the potential to differentiate into retina. Cells ahead of the MF express RD genes and anti-proneural genes to precisely control retinal cell fate determination and proneural cell differentiation. This region is referred to as the pre-proneural (PPN) domain, based on competence for retinal differentiation. The observation that the 2.8PB but not the 6.4BB enhancer os activated precociously in the PPN region suggests the presence of repressor elements residing within the 3.6BP fragment that contribute to the timing of atonal activation during MF progression. Interestingly, gain of function experiments in the wing disc did not reflect significant differences between 2.8PB and 6.4BB. Both enhancers conferred reporter expression only in groups of cells near the A/P compartment boundary in response to So and Eya and co-expression of dpp with so and eya led to an expansion of GFP expression mostly in the posterior domain. It is possible that some positive and negative factors required for the proper regulation of the ato 3′ enhancer in eye discs were not present in the wing disc. Previous studies have identified a number of genes sufficient to induce retinal tissue development or precocious photoreceptor differentiation, and these genes are potential candidates that contribute to the precise expression of ato. For example, ectopic expression of eyegone (eyg) or Optix (Optx) induces retinal tissue development while induction of mutant clones for either extradenticle (exd) or homothorax (hth) lead to ectopic eye formation in the ventral head region. Additionally, ectopic activation of the Hh signaling pathway or removal of hairy (h)/extramacrochaetae (emc) is sufficient to induce precocious furrow advancement and photoreceptor differentiation. Furthermore, removal of the Notch effector Su(H) causes slight advancement of neural differentiation. This search of conserved non-coding DNA sequences did not find predicted Ci binding sites in the ato 3′ cis-regulatory region. In contrast, a highly conserved transcription factor binding site for Su(H) is observed in the ato 3′ cis-regulatory region. Further analysis of ato 3′ eye enhancer should help to define the mechanisms that contribute to the precise control of its expression (Tanaka-Matakatsu, 2008).