clift/eyes absent
The Eyes absent proteins are members of a conserved regulatory network implicated in the development of the eye, muscle, kidney and ear. Mutations in the Eyes absent genes have been associated with several congenital disorders including the multi-organ disease bronchio-oto-renal syndrome, congenital cataracts and late-onset deafness. On the basis of previous analyses it has been shown that Eyes absent is a nuclear transcription factor, acting through interaction with homeodomain-containing Sine oculis (also known as Six) proteins. Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceeds by means of a thiol-phosphate intermediate. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Furthermore, the phosphatase activity of Eyes absent contributes to its ability to induce eye formation in Drosophila (Rayapureddi, 2003).
Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. The evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and a function is proposed for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation (Tootle, 2003).
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, 2000a).
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, 2000a).
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. The eya and so gene functions in the eye
appear strikingly similar -- not only in the adult, but also in
the developing eye disc.
This, together with functional synergy between the two in
eye formation, and a demonstrated physical interaction
between the two proteins in vitro supports the notion that
the two indeed function as (or in) a complex in eye formation.
Moreover, at the level of activation of this defined eya
enhancer, eya acts the same as eya and so together -- that is,
it fails to activate expression of the enhancer. That the
enhancer can restore eye development to eya mutants null
for early gene expression in the eye primordia also indicates that eya is not required for activation
of the enhancer. That directed expression of so fails to
induce ectopic eye formation in Drosophila may be due, in part, to the inability of so to activate
expression of the eya gene, an essential gene for eye
specification and a property shared by both ey and dac, both
of which can direct ectopic eye formation (Bui, 2000a).
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, 2000a).
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, 2000a).
Nk-2 proteins are essential developmental regulators from flies to humans. In Drosophila, the family member tinman is the major regulator of cell fate within the dorsal mesoderm, including heart, visceral, and dorsal somatic muscle. To decipher Tinman's direct regulatory role, a time course of ChIP-on-chip experiments was performed, revealing a more prominent role in somatic muscle specification than previously anticipated. Through the combination of transgenic enhancer-reporter assays, colocalization studies, and phenotypic analyses, two additional factors within this myogenic network were uncovered: by activating eyes absent, Tinman's regulatory network extends beyond developmental stages and tissues where it is expressed; by regulating stat92E expression, Tinman modulates the transcriptional readout of JAK/STAT signaling. This pathway is essential for somatic muscle development in Drosophila and for myotome morphogenesis in zebrafish. Taken together, these data uncover a conserved requirement for JAK/STAT signaling and an important component of the transcriptional network driving myogenesis (Liu, 2009).
This article presents a global map of the genomic regions bound by Tinman during multiple stages of mesoderm development, together with functional studies on the direct regulation and phenotypes of selected target genes. This analysis revealed several important features of Tinman's regulatory network. Although Tinman is primarily associated with its conserved role in heart development, many more target genes involved in somatic muscle development were identified. This implies that a major component of Tinman function is to orchestrate the transcriptional network driving early events in this tissue, including myoblast specification. In this context, Tinman's regulatory network is not only active in the dorsal mesoderm; the results pinpoint multiple nodes by which Tinman's regulatory influence extends to lateral and ventral regions of the embryo. First, Tinman directly regulates a number of identity genes essential for lateral and ventral muscle specification. For example, Tinman targets enhancers of slouch, which specifies two ventral muscle fibers (VT1 and VA3), apterous, which specifies three lateral and two ventral muscle fibers (LT1, LT2, LT3, and VA2, VA3), and ladybird (lbe, lbl), which specifies the lateral muscle fiber, SBM. Second, Tinman influences lateral and ventral muscle formation through the regulation of additional transcriptional cascades. A good example is eya, which is an integral component of the Tinman-regulatory network and is essential for the specification of somatic muscle in dorsal, lateral, and ventral regions of the embryo. Similarly, the data suggest that Tinman also directly regulates D-six4 and pox meso expression, two TFs essential for lateral and ventral somatic muscle development. In this manner, Tinman contributes to the general robustness of muscle specification, regardless of the muscle's position along the dorsal-ventral axis (Liu, 2009).
While examining the eya locus, an enhancer was identifed that fully recapitulates the spatiotemporal expression of eya within the mesoderm. This element is occupied by Tinman in vivo and requires tinman function for activity, indicating that Tinman provides direct input into eya expression via this eya-meso enhancer. Despite the strong dependence of the enhancer on tinman function, the expression of the endogenous eya gene is only marginally reduced in tinman mutant embryos. This result indicates a requirement for additional enhancers to regulate high-levels of eya expression within the mesoderm, one of which is most likely regulated by Twist. The expression of the stat92E gene in mesoderm is regulated in a similar manner. These types of regulatory connections (acting partially redundantly or for fine tuning), are often masked in genetics studies, yet serve as important inputs in generating robust regulatory networks (Liu, 2009).
The molecular nature of the ChIP-on-chip approach also uncovered a link between JAK/STAT signaling and muscle development, which was most likely masked in genetics studies due to the pleiotropic function of this pathway. Given the diverse cellular responses to this signaling cascade, including proliferation, apoptosis, and differentiation, the response of Stat activation in the context of myoblasts is currently not clear. A recent study on stat1 in C2C12 cells, a tissue culture model for myogenesis, suggests a potential role in proliferation. While this could partially explain the Drosophila muscle phenotype, it does not readily explain the defects in myotome boundary formation observed in zebrafish (Liu, 2009).
Although there are clear parallels between the role of eya-six genes and the JAK/stat pathway between flies and vertebrates, it is interesting to note that the positions of these genes within the overall myogenic network have diverged significantly. In vertebrates, Pax-Eya-Six act at the top of the transcriptional hierarchy, and are thus involved in the initiation of the myogenic network. In contrast, this regulatory module appears to function further down in the transcriptional hierarchy in flies. As a consequence, the upstream regulators of Eya-Six expression are not conserved. Similarly, as Nkx2 genes are not expressed in somites, stat5.1 cannot be regulated by these TFs in vertebrates, but is rather more likely to be regulated by members of the myogenic bHLH proteins, such as Myf5. Therefore, while the requirement of these key regulators is conserved, the wiring of these nodes within the overall network is highly diverged (Liu, 2009).
In summary, the systematic nature of this approach has revealed important regulators of myogenesis and partially redundant regulatory connections, both of which are often very difficult to uncover using standard genetic approaches (Liu, 2009).
clift/eyes absent is expressed within somatic gonad precursor cells (SGP) as these cells
first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in
gonadogenesis. In abdominal-A mutants, clift fails to be expressed, and in abd-A overexpressors, clift is likewise expressed ectopically. Despite the early expression of clift, SGP's
are specified in the absence of clift function. However, they fail to maintain
their fate; as a consequence, germ cells do not coalesce into a gonad. In
addition, using clift as a marker, it is shown that the anteroposterior and
dorsoventral position of the somatic gonadal precursor cells within a
parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with
a gene regulatory hierarchy involving abd A within the mesoderm (Boyle, 1997).
The Drosophila eyes absent gene is required for survival and differentiation of eye
progenitor cells. Loss of gene function in the eye results in reduction or absence of the adult
compound eye. Certain combinations of eya alleles undergo partial complementation, with dramatic
restoration of eye size. This interaction is sensitive to the relative positions of the two alleles in the
genome; rearrangements predicted to disrupt pairing of chromosomal homologs in the eya region
disrupt complementation. Ten X-ray-induced rearrangements that suppress the interaction obey
the same general rules as those that disrupt transvection at the bithorax complex and the
decapentaplegic gene. Moreover, like transvection in those cases, the interaction at eya depends
on the presence of normal zeste function. The discovery of transvection at eya suggests that
transvection interactions of this type may be more prevalent than generally thought (Leiserson, 1994).
To gain insight into the epistatic relationships among eyeless, sine oculis and eyes absent, their expression patterns in eye discs were compared. Ey expression in the eye disc starts in the embryo and is later observed in the entire eye disc of late second and early third instars. During subsequent development, Ey expression is strong in the region anterior to the furrow and downregulated in differentiating cells. Very little, if any, expression posterior to the furrow or in the region of the developing ocelli in third instar eye discs is detected with polyclonal antibody or by in situ hybridisation. At the furrow, the expression patterns of Ey and Decapentaplegic (Dpp) abut each other, indicating that Ey expression is downregulated just before cells enter the furrow. Eya and So start to be expressed in eye discs later than Ey. In contrast to Ey, neither So nor Eya is expressed in the eye anlagen of stage-16 embryos. Expression of Eya and So in the eye disc starts in the late second and early third instar, respectively. At these stages, both genes are expressed in a gradient with the strongest expression at the posterior of the eye disc. Later, when the furrow moves across the eye disc, So and Eya are expressed in a graded fashion with strongest expression just anterior to the furrow. In this region the expression pattern of Ey overlaps with those of So and Eya. However, in the most anterior part of the eye disc only Ey is detected at high levels. Unlike Ey, So and Eya continue to be expressed posterior to the furrow. Both genes are also expressed in the region of the differentiating ocelli. In summary, Ey is expressed in the eye disc from embryonic stages onward, until cells enter the furrow and start to differentiate, while So and Eya start to be expressed later, and cells begin to express increasing levels of So and Eya as the furrow moves across the eye disc. These results are consistent with ey acting upstream of so and eya during eye disc development (Halder, 1998).
Gene expression was studied in ey 2, so 1 and eya 1 mutant eye discs. Genetic and molecular data indicate that the so 1 and eya 1 alleles are amorphic or severely hypomorphic in the developing eye. Because massive cell death is observed in late third instar eye discs of all three mutants, gene expression analysis at this stage is not possible. Expression patterns were therefore studied in early third instar eye discs. At this stage all three genes are expressed and cells in the so 1 and eya 1 mutant eye discs are still viable. Eye discs from ey 2 mutants, however, already show first signs of morphological abnormalities, indicating that ey function is required prior to this stage. In eye discs of so 1 and eya 1 mutants, Ey is expressed normally, indicating that the functions of so and eya are not required for Ey expression. However, neither SO nor Eya expression is observed in ey 2 mutant eye discs. This demonstrates that ey function is required for eye disc expression of So and Eya. In about half of the so 1 mutant eye discs weak Eya immunoreactivity is detected, suggesting that so may not be required for EYA expression. Expression of So is not seen in eya 1 mutant eye discs. However, because So and Eya are expressed in nearly identical patterns and because both genes are required for cell viability, these results are not conclusive. In summary, (1) ey acts earlier than and upstream of so and eya in the developing eye disc and (2) the functions of so and eya in the eye disc appear to be dispensable for ey expression (Halder, 1998).
To further investigate the epistatic relationships among ey, so and eya, gene expression was examined in the developing extra eyes induced by Gal4-directed ectopic expression of eyeless. In wild-type third instar larvae So and Eya are not expressed in the wing disc proper. However, in wing discs that develop ey-induced extra eyes, both genes are ectopically expressed in and surrounding the developing photoreceptor clusters. These results indicate that ey acts upstream of so and eya during extra eye development. In order to investigate the dynamics and the spatial restriction of the induction of so and eya expression, ey was ubiquitously expressed in a temporally controlled manner using a heat-inducible transgene. Expression of so and eya was monitored by assaying lacZ expression of so and eya enhancer-traps. Ubiquitous expression of ey was induced starting at 83 hours after egg laying during the mid third instar stage. At this time neither so nor eya are expressed in the wing disc proper and eya is not expressed in leg discs. Two heat shocks induce only weak ectopic expression of so and eya; do not induce extra eye formation in adult flies, and just barely affect their morphology. This suggests that higher or prolonged levels of Ey may be required to efficiently reprogram cells into the eye developmental pathway. Consistent with this, induction of extra eyes is efficient when larvae carrying the heat-inducible ey transgene are heat-shocked six times. Such animals readily induce ectopic expression of so and eya; nearly 100% of pharate adult flies developed extra eyes. Although Ey is expressed ubiquitously, induction of both genes is confined to regions close to the A/P boundary that do not express Wg but do express Dpp. Thus, Ey alone is not sufficient to induce so and eya, bearing in mind that only those cells that are close to a source of Dpp appear competent to express so and eya in response to Ey. The finding that ey positively regulates so and eya transcription raised the possibility that so and eya may be required downstream of ey for ectopic eye formation. Indeed, targeted expression of ey is unable to induce ectopic eye development in so 1 and eya 1 mutant backgrounds, although ectopic Ey protein is produced and functional as inferred from its deleterious effects. Consistent with the lack of ectopic eye production, no ectopic photoreceptors develop in wing discs of so 1 and eya 1 mutants following targeted expression of ey (Halder, 1998).
Advantage was taken of the ectopic induction of so and eya by Ey to find out whether Ey activates so and eya in parallel and independently of one another or whether induction of one gene depends upon the function of the other one. The cell death phenotypes observed in the eye discs of so 1 and eya 1 make such an analysis difficult in the eye discs. It was reasoned that by expressing ey ectopically those requirements for cell viability might be bypassed. However, in late third instar larvae, ectopic Ey expression in so 1 and eya 1 mutant backgrounds causes ectopic cell death in wing discs and results in strongly reduced and deformed adult structures. Apparently, Ey is able to completely reprogram wing cells into the eye developmental pathway, even if that leads to cell death, as is the case in so 1 and eya 1 mutants. Nevertheless, in early to mid third instar wing discs, Ey induces ectopic expression of eya in a so 1 mutant background and, conversely, so is induced by Ey in an eya 1 mutant background. Therefore, both genes appear to be independent targets of Ey. However, the ectopic expression is weaker than that induced in a wild-type background, suggesting that so and eya are required for efficient induction of each other's expression. In summary, these results show that Ey acts upstream of so and eya and requires their function during ectopic eye induction (Halder, 1998).
In addition to its function in the developing compound eye, so is required for the formation of the entire visual system, including the optic lobes of the brain and the larval photoreceptor organs known as Bolwig's organs. In blastoderm-stage embryos, so is expressed in a dorsal domain of the head region that gives rise to those structures. Whether this region also includes the primordia of the eye discs is unknown and no so transcripts are detected in the eye discs when they become morphologically discernible toward the end of embryogenesis. A second Pax-6 gene has been isolated from Drosophila, designated twin of eyeless (toy), which is expressed in the developing head from the blastoderm stage onward. In contrast, ey starts to be expressed at germ band extension. The early expression of toy overlaps so expression in the head and their epistatic relationship has been investigated. Cytologically, toy maps close to ey on the fourth chromosome. Since no mutations in toy have been identified thus far, advantage was taken of a compound fourth chromosome to generate nullo 4 embryos that lack both toy and ey functions. Such embryos express so at normal levels in the head, indicating that toy is not required for so expression in the embryonic head. Similarly, toy is expressed in an appropriate pattern in embryos homozygous for a null allele of so. Therefore, so and toy appear to act in parallel during the development of the embryonic head of Drosophila. Later in development, so null embryos express toy and ey in the eye anlagen indicating that so is not only dispensable for that expression but also for the initial formation of the eye anlagen (Halder, 1998).
Retinal cell fate determination in Drosophila is controlled
by an interactive network of retinal determination (RD) genes, including eyeless, eyes absent, sine oculis and dachshund. The
role of decapentaplegic in this pathway was investigated.
During eye development, while
eyeless transcription does not depend on dpp
activity, the expression of eyes absent, sine oculis and
dachshund are greatly reduced in a dpp mutant
background. dpp signaling
acts synergistically with, and at multiple levels within, the retinal
determination network to induce eyes absent, sine oculis
and dachshund expression and ectopic eye formation. These
results suggest a mechanism by which a general patterning
signal such as Decapentaplegic cooperates reiteratively
with tissue-specific factors to determine distinct cell fates
during development (Chen, 1999).
During ectopic photoreceptor determination there is a tight correlation between
the location of ectopic eyes and the endogenous pattern of dpp
expression. In particular, the dpp-GAL4 driver is the most
efficient means of retinal induction by any of the RD genes: ubiquitous eyeless (ey) expression
induces downstream genes only in the vicinity of the
anteroposterior (AP) compartment boundary of discs where
dpp is normally expressed. These results
suggested that dpp signaling may be essential for the RD genes
to specify retinal cell fates. dpp is normally expressed along the AP boundary of the larval
wing disc. The GAL4 line 30A drives gene
expression in a ring that surrounds the wing pouch, which will
become the wing blade in the adult. The 30A
ring pattern corresponds to tissue that will form the hinge of
the adult wing and overlaps endogenous dpp at only two spots. When ey is misexpressed using 30A-GAL4, ectopic
eye formation is induced only at two positions: dorsal
and ventral to the pouch at the AP boundary. One explanation for this phenomenon is that dpp
activity is essential for ey to induce ectopic eye development.
Coexpression of dpp and ey
is sufficient to expand the domain of ectopic retinal
development induced by ey alone. To test whether dpp and ey act synergistically to induce RD genes, mRNA levels of ey, eya, so and dac were measured
in a dpp loss-of-function background. ey is normally expressed
throughout the entire eye disc prior to MF initiation and
anterior to the furrow during MF progression. In dpp mutants, the eye-antennal disc is much smaller than
in wild-type due to a proliferation defect, and MF initiation and
photoreceptor development does not occur. Nevertheless, EY mRNA is still
detectable in dpp mutant eye discs throughout second
and third instar larval development. In contrast,
although eya is still expressed in the ocellar region, almost no EYA, SO or DAC mRNA is detected in dpp mutant eye discs prepared from second or third
instar larvae. These data
indicate that dpp is not essential for ey expression but is
required upstream of eya, so and dac in the eye disc (Chen, 1999).
If eya and dac are the primary downstream targets of dpp
during eye development, then it should be possible to bypass
the requirement for dpp and induce ectopic eye formation by
overexpressing ey with eya or dac. While targeted expression
of either eya or dac alone driven by 30A-GAL4 is unable to induce
photoreceptor development, strong synergistic induction of
ectopic eye formation is observed when ey is coexpressed with
either dac or eya. Although there is clear synergy
between ey and dac or eya, ectopic photoreceptor induction in
both imaginal discs and adults is still limited to the vicinity of
the AP boundary and the source of dpp signaling. Moreover,
photoreceptor differentiation is still restricted to the vicinity
of the AP boundary when ey, dac, eya and so are
simultaneously induced by 30A-GAL4, indicating that dpp and
ey must regulate other essential targets in this process (Chen, 1999).
It is possible that dpp signaling might cooperate directly and
exclusively with ey. Alternatively, dpp could interact at multiple
levels within this pathway. To distinguish these two models, a
test was performed to see whether dpp functions synergistically with eya and so to
regulate the expression of dac. No ectopic dac expression is
induced by so alone: targeted expression of eya
induces ectopic dac expression only at a single ventral spot on
the AP boundary of the wing disc when driven by 30A-GAL4. Consistent with the idea that the Eya and So
proteins function cooperatively as a complex, strong synergistic induction of dac is observed when eya
and so are coexpressed. However, dac
expression is still restricted mainly to places where endogenous
dpp is present. In contrast, when dpp is coexpressed with eya,
strong dac expression is induced all along the ventral-posterior
pouch margin Moreover, ectopic Dac is detected
around the entire circumference of the wing pouch as a result of
dpp, eya and so coexpression. Since coexpression of dpp, eya and so is sufficient to
induce dac expression in places where dpp and ey cannot, it is
concluded that dpp interacts with the network at multiple levels to
control the expression of retinal determination genes. Consistent
with this interpretation, no induction of ey
transcription could be detected in response to misexpression of dpp, eya and so with
30A-GAL4 (Chen, 1999).
Thus dpp signaling is
reiteratively used to regulate gene expression within the retinal
cell fate determination pathway in Drosophila. Specifically, dpp signaling enables ey to induce strong eya,
so and dac expression in the posterior, but not anterior, wing
disc compartment. In contrast, dpp functions synergistically
with eya and so to activate the expression of dac in both
compartments. This activation of dac expression by dpp, eya
and so is unlikely to result from feedback induction of ey for two reasons: (1) targeted expression of ey and dpp
is unable to induce dac in the anterior wing disc compartment, and
(2) ectopic ey transcription is not detected in response to
misexpression of dpp, eya and so driven by 30A-GAL4 in the
wing disc. Thus, these data suggest that dpp signaling interacts
with the retinal determination pathway at (at least) two levels to
regulate RD gene expression. Interestingly, while
targeted expression of dpp, eya and so with 30A-GAL4 is
unable to induce ey expression or ectopic photoreceptor
development in the wing disc, coexpression of eya and so using
dpp-GAL4 is sufficient to induce ey expression and
photoreceptor development in the antennal disc. These differences most likely reflect the unique
transcriptional environments present in the specific portions of
each imaginal disc tested in these assays (Chen, 1999).
decapentaplegic mediates the effects of hedgehog in tissue patterning by regulating the expression of tissue-specific genes. In the
eye disc, the transcription factors eyeless, eyes absent, sine oculis and dachshund participate with
these signaling molecules in a complex regulatory network
that results in the initiation of eye development. Analysis of functional relationships in the early eye disc
indicates that hh and dpp play no role in regulating ey, but
are required for eya, so and dac expression. Ey is expressed throughout the eye portion of the wild-type
eye disc during early larval stages, prior to MF initiation. Eya and
Dac are expressed throughout the posterior half of the eye
imaginal disc, with stronger expression at the posterior margin. Ey is
expressed normally in homozygous Mad1-2 clones that touch
the posterior margin and in clones that are positioned internally in the disc,
indicating that Dpp signaling is not required for Ey expression
prior to MF initiation. In contrast, neither Eya nor Dac is
expressed in homozygous Mad1-2 clones that touch the margin
of the eye disc. In addition, Eya and Dac
are not expressed, or are expressed weakly, in internal clones
that lie well anterior of the posterior margin. However, strong Eya and Dac expression is observed in internal clones that lie within a few cell diameters of the posterior margin. Like Eya and Dac protein, SO mRNA is expressed in the
posterior region of the eye disc prior to MF initiation. Mad1-2 posterior margin clones fail to
express so. These results suggest that dpp
function is required to induce or maintain Eya, SO and Dac
expression, but not Ey expression, at the posterior margin prior
to MF initiation. This function is consistent with the pattern of
DPP mRNA expression along the posterior and lateral margins
at this stage of eye disc development. Whereas dpp is not necessary for Eya and Dac expression in internal, posterior regions of the early eye disc, it does play a role in regulating Eya and Dac expression in internal, anterior
regions of the disc. Although DPP mRNA
expression does not extend to the very center of the eye disc,
it is expressed in a significant proportion of the interior of the
disc. The possibility that dpp may regulate gene expression in
more central regions may be attributed to the fact that it
encodes a diffusible molecule (Curtiss, 2000).
Restoring expression of eya in loss-of-function dpp mutant
backgrounds is sufficient to induce so and dac expression
and to rescue eye development. Thus, once expressed, eya
can carry out its functions in the absence of dpp. These
experiments indicate that dpp functions downstream of or
in parallel with ey, but upstream of eya, so and dac.
Additional control is provided by a feedback loop that
maintains expression of eya and so and includes dpp. The
fact that exogenous overexpression of ey, eya, so and dac
interferes with wild-type eye development demonstrates
the importance of such a complicated mechanism for
maintaining proper levels of these factors during early eye
development. Whereas initiation of eye development fails
in either Hh or Dpp signaling mutants, the subsequent
progression of the morphogenetic furrow is only slowed
down. However, clones that are simultaneously
mutant for Hh and Dpp signaling components completely
block furrow progression and eye differentiation,
suggesting that Hh and Dpp serve partially redundant
functions in this process. Interestingly, furrow-associated
expression of eya, so and dac is not affected by double
mutant tissue, suggesting that some other factor(s)
regulates their expression during furrow progression (Curtiss, 2000).
The lack of eya, so and dac expression in Mad1-2 clones that lie at the margins of the eye disc prior to MF initiation reflects
a role for dpp in controlling early eye gene expression at these
stages of eye development. Evidence from several studies suggests that ey acts together with dpp at or
near the top of the hierarchy: (1) ey expression is not
regulated by dpp; (2) ey and
dpp are both required for eya, so and dac expression prior to
MF initiation;
(3) ey is not capable of rescuing dppblk eye development or of inducing ectopic eyes in regions of imaginal
discs in which dpp is not already expressed. These observations suggest that ey functions
upstream of or in parallel with dpp. The possibility that ey is
responsible for dpp expression, leading indirectly to eya, so and
dac expression, is unlikely. Since ey cannot induce ectopic eyes
without a source of dpp, it probably cannot induce dpp
expression, at least not in the absence of factors that are specific
to the eye disc. Moreover, Ey protein binds to the regulatory
region of so, suggesting it is directly
involved in so regulation. Thus, it is likely that ey and dpp
cooperate to induce expression of the other early eye genes (Curtiss, 2000).
Such cooperation could achieve two ends. (1) ey is
expressed throughout the eye disc and from embryonic stages
of development through MF initiation. However, induction of
eya, so and dac expression and MF initiation occurs
approximately 48 hours later, around the time of the transition
between second and third instars. Moreover, eya, so and dac
are not expressed throughout the eye disc as ey is, but have
stronger levels of expression around the margins than in other
regions. The initiation of dpp expression at the posterior
margin at approximately the same time suggests that it could
be the spatiotemporal signal that sets the MF in motion.
(2) dpp induces expression of tissue-specific genes as part
of its role in patterning many diverse structures in Drosophila.
An interaction with ey could be essential to ensuring that in the
eye imaginal disc dpp initiates factors that are appropriate to
eye development, such as eya, so and dac (Curtiss, 2000).
The function of the Dpp and Hh signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo has been analyzed. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknullt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain (Chang, 2001).
Dorsal epidermal and visual system fates, in particular those of the posterior optic lobe and larval eye, are not expressed in dpp loss of function. It is likely that these abnormalities are the result of changes in early head gene expression. This was followed in detail by assaying the expression of several regulatory genes known to be required for the normal development of the visual primordium, including otd, tll, so and eya in dpp-null mutants:
The observed downregulation of head gap genes and early eye genes in the dorsal midline is an indirect effect of Dpp mediated by the Dpp target zerknüllt (zen). Previous studies have demonstrated that high levels of Dpp in the dorsal midline upregulate and focus the expression of zen in the amnioserosa and, further anteriorly, in the dorsomedial head epidermis. An RNA in situ probe revealed the expression of zen in the early eye field of a stage 5-7 embryo. Assaying the expression of head gap and early eye genes in a zen-null mutant background demonstrates that Zen acts as a repressor of these genes. Whereas in wild type, after an initial unpaired expression straddling the dorsal midline, tll, so and eya are turned off in the dorsal midline, they continue to be expressed in this domain in a zen mutant. At later stages, lack of zen results in a cyclops phenotype (Chang, 2001).
In the head region, highest levels of Dpp are required to promote mid dorsal fates (head epidermis, analogous to amnioserosa in the trunk). The activation of screw is involved in this process, similar to its role in the dorsomedial trunk. Intermediate Dpp levels promote dorsolateral fates (visual primordium). Low levels of Dpp are reached in the protocerebral neurectoderm and are permissive for the formation of protocerebral neuroblasts. Several of the regulatory genes expressed in the anterior brain and eye field may be direct targets of Dpp signaling. The findings show that so, eya and omb are activated by Dpp in the visual primordium. These regulatory genes initiate the fate of visual structures, in particular larval eye and outer optic lobe. It has recently been shown that eya and so are also targets of Dpp signaling in the eye imaginal disc (Chang, 2001).
The secondary restriction of so (and other genes with bilateral expression domains developing from unpaired domains, including tll and otd) is effected by the Dpp target zen in the dorsal midline. This homeobox gene is expressed as a response to peak levels of Dpp in the dorsal midline, including amnioserosa and, in the head of the embryo, in the dorsomedial head epidermis primordium. Loss of zen, similar to reduction of Dpp, results in the absence of amnioserosa and head epidermis, and a cyclops phenotype (Chang, 2001).
In view of these results, it is speculated that the interaction between Dpp and Hh is indirect and requires the function of so, eya and possibly other 'early eye genes' -- according to this model, Dpp activates so and eya in the eye field. Slightly later, expression of so and eya is lost dorsomedially, due to repression by Zen at this level. In a second step, the expression of Hh (which comes on later than Dpp) triggers larval eye fate in cells close to the Hh source. The response of a cell to Hh, that is, its expression of ato, depends on its previously expressing so and eya. Finally, Ptc inhibits the range of Hh action, similar to its alleged function in the trunk and imaginal discs (Chang, 2001).
A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:
In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior
(P) to the anterior (A) of the eye imaginal disc. Several subdomains along the A-P axis of the eye disc have been described that express distinct
combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and
homothorax (hth), and the zinc-finger gene teashirt (tsh). Evidence suggests that this combination of transcription factors may
function as a complex and that their combination plays at least two roles in eye development: it blocks the expression of later-acting transcription factors
in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Dpp (Bessa, 2002).
Anterior to the MF, at least three cell types can be distinguished
by the patterns of Hth, Ey, and Tsh expression. The most anterior domain in the eye field, which is next to the antennal portion of the eye-antennal
imaginal disc, expresses Hth, but not Tsh or Ey. In a
slightly more posterior domain, all three of these factors are
coexpressed (region II). In a more posterior domain, Tsh and Ey, but not Hth, are coexpressed. This domain, which also expresses hairy, is equivalent to the pre-proneural (PPN) domain. The MF, marked by the expression of Dpp, is immediately posterior to the PPN domain, and therefore abuts Tsh + Ey-expressing cells (Bessa, 2002).
Domain II is the only region of the eye-antennal imaginal disc that
strongly expresses all three of these transcription factors. Posterior
to the MF, Hth, but not Tsh or Ey, is expressed in cells committed to
become pigment cells. Hth and Ey, but not Tsh, are coexpressed in a
narrow row of margin cells that frame the eye field and separate the
main epithelium of the eye disc from the peripodial membrane. Finally, Hth is also strongly
expressed in peripodial cells, whereas Ey and Tsh are weakly expressed
in a subset of these cells (Bessa, 2002).
The expression patterns of So, Dac, and Eya were also examined in
wild-type eye discs. All three of these transcription factors are
expressed in the PPN domain but not in domain II. Their expression domains have the same anterior limit but different posterior limits. Furthermore, the anterior limits of their expression domains are not sharp, but instead decrease gradually as Hth levels increase. Thus, cells in the PPN
domain express So, Dac, and Eya as well as Tsh, Ey, and Hairy. Anterior
to the PPN domain there is a gradual transition into domain II, where
cells express Hth, Ey, and Tsh, but not So, Eya, Dac, or Hairy (Bessa, 2002).
The complementary patterns of Hth versus So, Eya, and Dac at the
transition between domain II and the PPN domain suggested that these
factors may also be playing a role in hth repression. To test
this idea, clones of cells mutant for eya were examined. eya- clones de-repress hth. Part of this de-repression is probably due to the fact that dpp expression requires eya. However, the de-repression of hth is
observed in all eya- cells, even in cells that are
next to wild-type, dpp-expressing cells. Thus, Dpp
expressed in wild-type neighboring cells is not able to repress
hth in adjacent eya- cells. These data
suggest that eya is required for Dpp to repress hth
in the PPN domain. hth was also de-repressed in
dac- clones, suggesting that
dac also plays a role in hth repression (Bessa, 2002).
Because Hth is coexpressed and can interact in vitro with Tsh and Ey,
the possibility was considered that combinations of these transcription
factors might be required to repress eya and dac. Consistent with this idea, it was found that the simultaneous expression of
Tsh and Hth efficiently represses eya and dac
expression. Importantly, the dual expression
of Tsh and Hth is maintained by Ey expression; consequently, these clones expressed all three of these transcription factors. Other pairs of
these transcription factors (Hth + Ey and Tsh + Ey) were also tested, and it was found that
they can also partially repress eya (Bessa, 2002).
The above results suggest that the combination of Hth + Ey + Tsh, which is normally present in domain II, is able to repress the expression of eya. To test if hth normally plays a role in the repression of these genes,
hth- clones were examined. Although hth-
clones anterior to the MF are rare, it was found that both
dac and eya are de-repressed in anterior
hth- clones (Bessa, 2002).
In summary, these data suggest that the combination of the factors
expressed in domain II is necessary and sufficient to repress eya and dac. In contrast, Hth is sufficient to
repress the pre-proneural gene hairy. Conversely, eya
and dac, together with Dpp, repress hth as the MF
advances. It is suggested that one function for this reciprocal antagonism
may be to prevent premature and uncoordinated differentiation anterior
to the MF. However, as the MF advances, hth must be repressed
to allow differentiation to occur (Bessa, 2002).
These experiments suggest that one of the functions mediated by
Ey-Hth-Tsh is to repress eya and dac. This
proposal stems from both ectopic expression experiments, showing that
the coexpression of Ey, Hth, and Tsh represses these genes, and from loss-of-function experiments, showing that hth-
clones anterior to the MF de-repress these genes. Similarly, hth is de-repressed in both eya- and
dac- clones, suggesting that this antagonism exists
in both directions. Interestingly, the antagonism between these two
sets of genes is analogous to that observed in other appendages. In the
leg, hth and tsh are required for the development of
proximal fates, and have been shown to be mutually antagonistic with
dac and Distal-less (Dll), two genes
required for intermediate and distal leg fates, respectively. Similarly, in the
wing, hth and tsh are required for proximal wing
fates, and oppose the activity of vestigial (vg),
which is required for more distal wing fates (Bessa, 2002).
The Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling
establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye
specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of
these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is
complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).
In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin,
or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless,
suggests a possible function for Wingless in the regulation of the eye
selector genes. The top of the genetic hierarchy involved in eye specification
appears to be the Pax6 homolog, Eyeless. In the
third instar eye disc the expression of Eyeless is restricted to the region
anterior to the furrow and, despite the Wingless-induced inhibition of eye
development, the expression of Eyeless in this region is not affected by
axin- clones. This lack of an effect anterior to the furrow,
despite the overgrowth and abnormal Distal-less expression in the same region,
implies that misregulation of Eyeless is not the primary cause of the
transformations caused by ectopic Wingless activity (Baonza, 2002).
Downstream of Eyeless (although feedback relationships makes the epistatic
relationship complex) are other transcription factors required for eye
specification, including Eyes absent, Sine oculis and Dachshund. A
phenotype similar to axin- clones of excess proliferation
and consequent overgrowth is caused by loss of Eyes absent and Sine oculis.
Moreover, as in axin- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).
The expression patterns of Eyes absent, Sine oculis and Dachshund, in axin- and/or arm* mutant clones, were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin- M+ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin- M+ clones. Consistent with these results, in arm*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).
The conclusion that Wingless signaling negatively regulates the expression
of Eyes absent, Dachshund and Sine oculis anterior to the furrow leads to the
prediction that in normal development, domains of high Wingless activity in
the anterior region of the eye disc will be associated with low expression of
these genes. Previous work indicates that their expression is broadly
non-overlapping, but to analyse this precisely, discs were double-labelled
to detect the expression of Wingless and Eyes absent or Sine oculis throughout
the third instar larval stage. The expression of these eye specification genes
is precisely complementary to that of Wingless in the anterior lateral margins
of the eye throughout the third instar. This is consistent
with a role for Wingless signaling in initiating the borders between eye and
other head structures. Note that in posterior lateral regions
slight overlap is observed between the expression of Wingless and these genes; this is presumably analagous to the expression of eye specification genes seen in some posterior axin- clones, and confirms that in
posterior regions of the eye disc, Wingless signaling is not incompatible
with the expression of these genes (Baonza, 2002).
These results indicate that Wingless regulates the
final size of the eye field of cells by controlling the expression of eyes
absent, sine oculis and dachshund. The expression pattern of
these genes in the anterior eye margin is complementary to the expression of
Wingless throughout the third instar, indicating that in anterior regions,
high activity of Wingless signaling corresponds to absence of these gene
products. Moreover, ectopic activation of Wingless signaling represses their
expression anterior to the furrow (where they act to specify the eye field)
throughout eye development. Finally, the loss of Wingless signaling causes
ectopic expression of Eyes absent and Dachshund (Baonza, 2002).
It is proposed that the initial expression of Eyes absent, Sine
oculis and Dachshund is negatively regulated by Wingless signaling in the eye
disc, and that this regulation initiates the border between the eye field and
adjacent head cuticle. Attempts were made to define whether Wingless represses
the eye specification genes independently or whether eyes absent is
the primary target but the data confirms earlier reports of the complexity of
the regulatory relationships between eyes absent, sine oculis and
dachshund. The observation that Eyes absent is able partially to
restore the expression of the other two genes but cannot rescue the overgrowth
and differentiation phenotype of axin- clones has two
possible explanations. Either Wingless represses eye development through at
least one additional gene, or high level Wingless signaling blocks eye
development later in the developmental program -- e.g., it is known to inhibit
morphogenetic furrow initiation, even after its earlier effects are rescued by
eyes absent expression (Baonza, 2002).
Although Hedgehog (Hh) signaling is essential for morphogenesis of the Drosophila eye, its exact link to the network of tissue-specific genes that regulate retinal determination has remained elusive. In this report, it is demonstrated that the retinal determination gene eyes absent (eya) is the crucial link between the Hedgehog signaling pathway and photoreceptor differentiation. Specifically, it is shown that the mechanism by which Hh signaling controls initiation of photoreceptor differentiation is to alleviate repression of eya and decapentaplegic (dpp) expression by the zinc-finger transcription factor Cubitus interruptus (Cirep). Furthermore, the results suggest that stabilized, full length Ci (Ciact) plays little or no role in Drosophila eye development. Moreover, while the effects of Hh are primarily concentration dependent in other tissues, hh signaling in the eye acts as a binary switch to initiate retinal morphogenesis by inducing expression of the tissue-specific factor Eya (Pappu, 2003).
Misexpression of eyeless (ey) in the wing disc causes ectopic photoreceptor
differentiation only in regions where both dpp and hh
signaling are normally active. The simplest explanation for this effect
invokes a linear regulatory hierarchy where hh induces dpp, which in turn cooperates with ey to initiate retinal morphogenesis. While misexpression of ey and dpp together does indeed lead to synergistic photoreceptor differentiation, this occurs only in the posterior compartment of the wing disc. Notably, Hh signaling is not transduced in the posterior compartment of the wing disc due to the repression
of ci by En. Furthermore, dpp and ey expression does not induce Ci expression in the posterior compartment of the wing disc. Thus, it is concluded that dpp and ey can induce Eya expression and photoreceptor differentiation in the posterior compartment of the wing disc in
the absence of Hh signaling and Cirep. Misexpression of hh and ey induces robust eya expression and photoreceptor differentiation in the wing disc, but only in the anterior compartment. This result is consistent with a model in which Hh signaling normally blocks the
production of Cirep and converts it into an activated form,
Ciact, in the anterior compartment of the wing disc.
Ciact can induce dpp expression in the anterior
compartment and dpp can in turn cooperate with ey to
induce robust Eya expression and photoreceptor differentiation. Consistent with this model, co-expression of hh, dpp and ey leads to Eya expression and photoreceptor differentiation in both compartments of the wing disc. Taken together, these results suggest that, in the wing disc, ey and dpp can activate eya expression only in the absence of Cirep (Pappu, 2003).
Co-expression of dpp, ey and eya using the
30A-Gal4 driver induces photoreceptor differentiation in both wing compartments, albeit with low penetrance. This effect becomes stronger and more penetrant when dpp, ey, eya and so are misexpressed in
a ring around the wing pouch. These results demonstrate that providing ey, dpp and eya from an exogenous source is sufficient to bypass the requirement for Hh signaling during initiation of ectopic photoreceptor differentiation. In addition, these results implicate eya as a key
target for Hh signaling during the initiation of normal retinal morphogenesis, most likely by blocking Cirep (Pappu, 2003).
The data from ectopic expression analyses in the wing disc suggest that Cirep has a major role in blocking eya expression in areas that are not exposed to Hh signaling. However, Ciact also plays an important role in patterning the anterior compartment of the wing disc. For
example, adult wings that contain ci mutant clones develop with
defects in the anterior compartment. In the Drosophila eye disc, ci is expressed uniformly but Ci protein expression follows a dynamic pattern. It has been proposed that in regions anterior to the furrow Ci is subject to PKA-dependent phosphorylation and SCFSlimb-dependent processing into Cirep. Cells in the MF, however, receive and transduce the Hh signal and prevent the proteolytic processing of Ci, therefore blocking production of Cirep. Furthermore, it has been proposed that cells that are posterior to the MF do not accumulate Cirep in a PKA-dependent manner. Instead, these cells use a smo- and cullin3- dependent proteolytic process leading to the complete degradation of Ci. Therefore, the role for Ci in the eye appears to be limited only to cells that are part of, and anterior to, the MF. However, these studies do not establish separate functional roles for Ciact and Cirep in the developing eye (Pappu, 2003).
Surprisingly, Eya expression and photoreceptor differentiation are not perturbed in Drosophila eye discs that contain large ci-null mutant clones. Similarly, adult eyes containing large ci mutant clones appear normal both externally and in internal
sections. These results, coupled with ectopic expression analysis in the
wing disc, suggest that Ciact plays little or no role during normal photoreceptor differentiation. Furthermore, these results support a model in which the major role for Hh signaling during the initiation of photoreceptor differentiation is to prevent the production of Cirep (Pappu, 2003).
Interestingly, ci-null mutant clones that span the furrow do not hasten furrow progression. Although ectopic activation of the Hh pathway is sufficient to induce precocious furrow advancement and photoreceptor differentiation, loss of Ci is not. A likely explanation for this apparent contradiction may be found in the distinction between loss- and gain-of-function experiments. Specifically, although Ciact normally plays little or no role in eye development, ectopic production of Ciact is sufficient to induce precocious furrow advancement. Intriguingly, vertebrate homologs of Drosophila ci have evolved to
carry out either activator (Gli1 and Gli2) or repressor
(Gli3 and perhaps Gli2) functions independently.
These findings demonstrate that in the absence of gene duplication,
tissue-specific separation of these functions has also occurred in
Drosophila (Pappu, 2003).
It is proposed that Hh signaling acts as a binary switch during
Drosophila eye development to control the timing of initiation of
photoreceptor differentiation. Specifically, the data suggest that during early larval development Cirep normally inhibits retinal morphogenesis by blocking eya and dpp expression. Hh signaling in late second instar larvae blocks production of Cirep, which in turn allows dpp and eya expression, MF initiation, progression and photoreceptor differentiation. Rather than regulating the
differentiation of multiple cell types in a concentration-dependent manner, the data suggest that Hh signaling acts as a molecular switch that is sufficient to initiate dpp and eya expression and retinal morphogenesis. This model also explains the seemingly contradictory phenotypes of loss of smo (blocks MF initiation) and loss of ci (no effect) during Drosophila eye development. Loss of ci creates a permissive environment for eya and dpp expression and photoreceptor differentiation, rendering eye development Hh independent.
By contrast, Cirep persists in the absence of smo function and thus photoreceptor morphogenesis does not occur in smo clones. Since ci null mutant clones in the eye develop normally, other Hh independent mechanisms must also act to control the initiation of retinal morphogenesis in Drosophila (Pappu, 2003).
Posterior margin smo mutant clones lack Eya expression and
photoreceptor differentiation. The lack of eya
expression in these cells is attributed to their inability to block the production of Cirep. Furthermore, the data demonstrate that co-expression of dpp and eya in these posterior smo mutant clones rescues photoreceptor differentiation. In addition, dpp and eya co-expression is sufficient to rescue delayed furrow progression in smo clones. However, the precise temporal and spatial order of photoreceptor recruitment may not be rescued in these clones. Thus, the requirement for Hh signaling in the eye can be circumvented by the expression of the downstream targets dpp and eya. These results demonstrate that eya is a crucial eye-specific target of Hh signaling during the initiation of retinal differentiation and has led to a new model
for the initiation of retinal morphogenesis. In this model, Hh
signaling blocks the proteolytic degradation of Ciact into
Cirep, thus allowing initiation of dpp expression. Once dpp expression is established, the absence of Cirep allows dpp to act in parallel with ey to initiate eya expression, which in turn leads to so expression. Furthermore, dpp cooperates with eya and so to initiate the expression of dac and extensive feedback regulation among these genes leads to consolidation of retinal cell fates (Pappu, 2003).
The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). The function of these signaling pathways in this process has been investigated. Organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. It is proposed that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development (Kenyon, 2003).
This paper analyzes the role of Dpp and Notch in the regional specification of the eye-antennal disc. This study makes four observations: (1) domains of regional identity emerge in a complex pattern starting early in L2; (2) formation of eye and antenna primordia depend upon specific domains of dpp expression that emerge in early-L2 (eye) and mid-L2 (antenna); (3) neither Notch nor Dpp control the establishment of separate eye and antennal fields; (4) Notch can influence the establishment of an eye primordium through its control of proliferation in the eye field. Current models of regional specification have been evaluated based on these results and a new perspective on the emergence of regional identity in this tissue is presented (Kenyon, 2003).
It has been proposed that allocation of eye field and antennal field identity occurs in the latter half of L2 through the restriction of eye selectors, such as Ey, and antennal selectors, such as Dll, to distinct regions of the disc. However, two observations reported in this paper are not consistent with this interpretation: (1) Dll is not expressed ubiquitously at any time during disc development; (2) eye and antennal fields are clearly established by mid-L2 as evidenced by the restricted expression of Ey (eye field) and Cut (antennal field), and by distinct Dpp/Wg patterning centers within each field. These observations place the emergence of separate eye and antennal fields in the first half of L2 and not in the second half as previously proposed. Moreover, onset of Eya occurs in early-L2 and so is expressed by mid-L2. The beginning of eye primordium formation in early-L2, prior to the appearance of distinct fields, indicates that regional specification within this disc does not follow a two-step mechanism (i.e., establishment of separate fields followed by induction of organ primordia) but occurs in a more complex pattern. Further analysis of the transcription factors and signaling molecules active in the late-L1 and early-L2 disc is necessary to better understand how the establishment of eye field identity relates to eye primordium formation and the emergence of an antennal field (Kenyon, 2003).
Analyses of hypomorphic dpp alleles and tissue mutant for Mad implicate Dpp in the control of eya and Dll expression during late larval development. The onset of Eya and Dll expression correlate with specific changes in dpp expression during normal development. Using temperature shift experiments, it has also been established that Dpp signaling in L2 is required for the proper induction of both Eya and Dll in their respective fields. Gain-of-function analyses show that Dpp is also sufficient to induce Eya expression within the eye field and Dll expression within the antennal field. Clearly, though, Dpp must function in the context of selector factors such as Ey in order to produce two independent primordia within the eye-antennal epithelium. In the presence of Ey, Dpp signaling induces Eya expression as opposed to Dll. The absence of Ey in the antennal field at the time of Dpp signaling is of crucial importance to ensure the proper induction of Dll and subsequent formation of an antenna primordium. Indeed, as described in this study, the restriction of Ey to the eye field precedes the emergence of dpp and Dll in the antennal field during normal development (Kenyon, 2003).
In conclusion, Dpp, unlike Notch, functions as an inducer of tissue identity during specification of the eye-antennal disc, and the spatial and temporal aspect of organ primordia formation is controlled at the level of dpp transcription (Kenyon, 2003).
The effect of field size on eye primordium formation cannot be simply mediated by Dpp but is likely due to the influence of a third signaling system, the Wingless pathway. Wg functions as a negative regulator of eye development and is known to antagonize Dpp signaling in L3 discs. This antagonistic interaction occurs at least in part at the posttranscriptional level and is likely established earlier in development. At the time of onset of Eya expression, early in L2, the sources of dpp and wg are localized to opposing regions of the eye-antennal disc -- dpp along the posterior margin and wg across the dorsal anterior region. Hence, the relative concentration of Dpp and Wg experienced by disc cells likely depends on their location within and the size of the morphogenetic field. Since Dpp induces Eya expression and Wg antagonizes Dpp signaling, field size becomes a critical variable in determining the response to Dpp/Wg signaling and thus influences eye primordium formation (Kenyon, 2003).
This model readily accounts for the changes in Eya expression observed in the various genetic backgrounds. In discs expressing Notch antagonists, dpp and wg are still expressed; however, inhibition of cell proliferation results in a smaller disc and a smaller morphogenetic field. This reduction in size changes the balance between Dpp and Wg signaling resulting in a lack of Eya induction. In this context, stimulation of cell proliferation by CycE increases field size, thus restoring relative levels of Dpp and Wg signaling compatible with Eya induction. A simple prediction of this model is that modification of Dpp/Wg signaling in favor of Dpp should restore Eya expression in small ey-Gal4 UAS-SerDN discs. This was tested by removing one wild-type copy of the wg gene and thus lowering Wg signaling in SerDN-expressing discs. In agreement with the model, Eya expression is significantly rescued in late-L2/early-L3 wg+/+ey-Gal4 UAS-SerDN discs regardless of disc/field size (Kenyon, 2003).
The Drosophila ovary provides a model system for studying the mechanisms that regulate the differentiation of somatic stem cells into specific cell types. Ovarian somatic stem cells produce follicle cells, which undergo a binary choice during early differentiation. They can become either epithelial cells that surround the germline to form an egg chamber ('main body cells') or a specialized cell lineage found at the poles of egg chambers. This lineage goes on to make two cell types: polar cells and stalk cells. To better understand how this choice is made, a screen was carried out for genes that affect follicle cell fate specification or differentiation. extra macrochaetae (emc), which encodes a helix-loop-helix protein, was identified as a downstream effector of Notch signaling in the ovary. Emc is expressed in proliferating cells in the germarium, as well as in the main body follicle cells. Emc expression in the main body cells is Notch dependent, and emc mutant cells located on the main body fail to differentiate. Emc expression is reduced in the precursors of the polar and stalk cells, and overexpression of Emc caused dramatic egg chamber fusions, indicating that Emc is a negative regulator of polar and/or stalk cells. Emc and Notch are both required in the main body cells for expression of Eyes Absent (Eya), a negative regulator of polar and stalk cell fate. It is proposed that Emc functions downstream of Notch and upstream of Eya to regulate main body cell fate specification and differentiation (Adam, 2004).
Molecular epistasis indicates that Notch signals through Emc to induce or
maintain Eya expression in the main body follicle cells. Eya expression is
lost in large Notch mutant follicle cell clones and can be induced by
forced expression of activated Notch in the follicle cells. Eya is involved in
inhibiting polar and stalk cell fate, so one might expect that Notch
or emc loss-of-function mutants would make extra polar cells. This is
not the case, however; it seems likely that the reason Notch and
emc mutant cells do not become polar cells is that they fail to
differentiate. Eya does not appear to be required for differentiation, but
rather for main body cell fate, since eya mutant cells differentiate
into polar cells. Thus, the Notch pathway must branch downstream of Emc, with
one pathway leading to Eya expression and repression of polar cell fate, and a
separate pathway leading to differentiation (Adam, 2004).
Emc can have both positive and negative effects on the number of polar
cells. Although this might initially seem mysterious, it can be explained by
the dynamic expression of Emc in ovaries. Emc is expressed in the germarium,
but it is reduced in polar/stalk precursors. Its expression remains low in
stalk cells but, in polar cells, returns to the same level as that of their
neighbors around the time of differentiation. Temperature-shift
experiments show that forced expression of Emc in immature polar cells can
lead to expression of Eya, which is the presumed cause of loss of polar cells.
By contrast, forced expression of Emc in maturing polar cells appears to lead
to potentiation of polar cell number, i.e., polar cell number per group is not
reduced from four to two, as in wild-type, but remains at four polar cells per
group. This is probably due to a role for Emc in polar cell differentiation,
because loss-of-function emc clones can result in loss of polar
cells (Adam, 2004).
Three lines of evidence suggest that Emc may be a key regulator of Eya
expression. (1) Emc and Eya expression are similar in multiple respects.
Both are upregulated in follicle cells in region 2B of the germarium, and in
main body follicle cells from stages 2 through 6. Both are downregulated in
the polar/stalk lineage from the germarium through stage 3, and in the
oocyte-associated follicle cells at stages 7 through 9. (2) Emc is
required for Eya expression in the main body. (3) Emc and Eya produce
similar overexpression phenotypes, including fused egg chambers and the loss
of polar cells, and, when Emc is overexpressed in the polar cells, Eya
expression is induced. Taken together, these data suggest that the expression
of Eya in the follicle cells is largely regulated by Emc. Emc is a
helix-loop-helix protein that lacks the basic DNA-binding domain of the bHLH
transcription factors. It normally opposes the activity of bHLH transcription
factors by sequestering them in non-productive complexes. Thus, the dependence
of Eya on Emc is likely to be indirect. Presumably, Emc inhibits a bHLH
protein that inhibits expression of Eya. The identity of this protein remains
an interesting subject for further study (Adam, 2004).
In Drosophila, the eye primordium is specified as a subdomain of the larval eye disc. The Zn-finger transcription factor teashirt (tsh) marks the region of the early eye disc where the eye primordium will form. Moreover, tsh misexpression directs eye primordium formation in disc regions normally destined to form head capsule, something the eye selector genes eyeless (ey) and twin of eyeless (toy) are unable to do on their own. Evidence suggests that tsh induces eye specification, at least in part, by allowing the activation of eye specification genes by the wingless (wg) and decapentaplegic (dpp) signaling pathways. Under these conditions, though, terminal eye differentiation proceeds only if tsh expression is transient (Bessa, 2005).
In order to test if tsh is sufficient to induce eye primordium identity in PE cells, the expression of the eye selector gene ey, as well as that of the early retinal genes eya and Dac, was examined in tsh-expressing clones. tsh-positive cells show increased Ey expression. In addition, PE tsh-expressing clones that lie close to the posterior margin activate eya and the eya target Dac, indicating that these cells adopt an eye primordium-like fate. PE clones overexpressing ey are not able to induce eya, neither are similar toy-expressing clones, in which ey expression is upregulated. In these PE clones, tsh expression is not induced. Therefore, it is concluded that neither ey upregulation nor the joint overexpression of toy and ey are able to re-specify the peripodial epithelium. In addition, overexpression of eya in PE clones do not turn Dac on either, which reinforces the idea that PE re-specification as eye primordium occurs only if tsh is expressed (Bessa, 2005).
Expression of tsh activates eya expression mostly in the center and posterior half of the PE, but not in the anterior half. Clones in this anterior region retain the expression of hth, which is normally expressed in all PE cells. Since dpp and wg are expressed in the domains of the posterior and anterior discs, respectively, it was reasoned that these differences in the response of tsh-expressing cells could be the result of these signaling pathways acting differently in anterior and posterior domains of the PE (Bessa, 2005).
To test this hypothesis, the response of normal PE cells to variations in both wg and dpp pathways was tested. Clones where the dpp pathway was hyperactivated through the expression of a constitutively active dpp-receptor, thick veins (tkvQD), or blocked by removing the signal transducer Mothers against dpp (Mad), showed no induction of eya expression or cell morphology changes. Neither did anterior clones expressing Axin, a negative regulator of the wg pathway or overexpressing wg. Nevertheless, when alterations in the dpp and wg pathways were performed in the presence of ectopic tsh, PE cells showed gene expression responses characteristic of the ME. Thus, whereas posterior tsh-expressing PE cells induce eya expression, tsh-expressing cells in which the dpp pathway has been blocked by removing Mad no longer express eya. Again, this is the behavior exhibited by tsh+ ME cells deprived of dpp signaling. Similarly, while anterior tsh-expressing PE cells retain hth expression, most clones expressing both tsh and Axin lose hth expression, as they do if Axin is expressed in the ME within the tsh domain. PE tsh+ tkv+ clones still fail to activate eya in anterior dorsal and anterior ventral regions, suggesting that even in these clones wg signaling can prevent PE re-specification. Clones of PE cells expressing tsh, tkvQD and Axin now activate eya anywhere in the disc, indicating that, in the presence of tsh, wg and dpp antagonize each other to regulate eya expression. It is noted, however, that the squamous to columnar cell shape change induced by tsh is independent of the activity of the wg and dpp pathways. These results suggest that tsh, when expressed in the PE, can reprogram this epithelial layer to respond to wg and dpp signals such that it develops in an eye primordium-specific manner (Bessa, 2005).
During the development of the eye disc, only cells of the ME will be specified as eye primordium. Although Wg and Dpp signals play essential roles during eye development, PE cells are relatively insensitive to these signaling pathways, as measured by cell survival, morphology, proliferation or gene expression changes. tsh starts being expressed in the ME around the time when the eye primordium is specified, and tsh has the potential to redirect eye disc PE cells towards eye development, an ability the eye selector genes toy and ey do not have on their own. These results indicate that the PE can be re-specified by tsh throughout most of the life of the larva. Thus, tsh-expressing clones induced during L1 and L2 induce eya and Dac expression. The transient expression of tsh during L2, or its induction by Gal4 drivers active during late-L2/L3, results in ectopic PE eyes (Bessa, 2005).
How organ identity is determined is a fundamental question in developmental biology. In Drosophila, field-specific selector genes, such as eyeless (ey) for eyes and vestigial (vg) for wings, participate in the determination of imaginal disc-specific identity. Gain-of-function screening was performed and a gene named winged eye (wge) was identified, that encodes a bromo-adjacent homology domain protein that localizes at specific sites on chromosomes in a bromo-adjacent homology domain-dependent manner. Overexpression of wge induces ectopic wings with antero-posterior and dorso-ventral axes in the eye field in a region-specific Hox gene- (Antennapedia) independent manner. Overexpression of wge is sufficient for ectopic expression of vg in eye discs. A context-dependent requirement of wge was demonstrated for vg expression in wing discs and for expression of eyes absent (eya), a control gene for eye development downstream of ey, in eye discs. In contrast to vg, however, overexpression of wge inhibits Eyeless-mediated expression of eya. Consistent with colocalization on polytene chromosomes of Wge and Posterior sex combs (PSC -- a Polycomb group gene product), an antagonistic genetic interaction between wge and Psc was demonstrated. These findings suggest that wge functions in the determination of disc-specific identity, downstream of Hox genes (Katsuyama, 2005).
Artificial activation of Notch signaling induces various ectopic appendages, such as ectopic eyes, antennae, wings, and legs, in the eye field in a context-dependent manner. In this system, the ey enhancer-dependent activation of Notch signaling and gene expression is crucial for transdetermination within a limited time window and for shutting off the forced expression once the transdetermination is induced. To identify the genes capable of changing disc-specific identity, a system with a P element-based GS vector was used. For a pilot experiment, 106 lines harboring the GS vector (GS lines) were crossed with flies carrying the ey enhancer-GAL4 (ey-GAL4) and a construct for the constitutively active Notch receptor under an upstream-activating sequence for GAL4 (UAS-Nact). Ectopic structures were induced in the eye field in combination with Notch signaling activation in five lines. For example, ectopic wings were induced in GS 1068 in which ey-GAL4 drives the expression of PGRP-LE, CG8509, and sd, encoding a cofactor of Vg. In the absence of Notch signaling activation, all five lines had reduced eye phenotypes. Therefore, to increase the screening efficiency, a prescreening step was introduced in which GS lines were crossed with the ey-GAL4 driver, and the resulting lines with reduced eye phenotypes were crossed with the ey-GAL4, UAS-Nact line. To confirm the effects of the prescreening, 74 negative GS lines were crossed with normal eyes in a prescreening with an ey-GAL4, UAS-Nact line. None of the lines had ectopic structures in the eye field, indicating that the prescreening was effective. In the prescreening, 8,486 lines had normal eyes and 1,202 lines had reduced eye phenotypes. In 9 of 9,710 lines, ectopic structures were induced in the eye field, e.g., ectopic wings in the GS 15923, even in the absence of Notch signaling activation. The resulting 729 lines with reduced eye phenotypes or ectopic structures in the eye field in the prescreening and 22 lines that were selected without prescreening were then crossed with the ey-GAL4, UAS-Nact line, and ectopic structures were induced in 45 lines in combination with Notch signaling activation (wing, 3 lines; antenna, 26 lines; leg, 16 lines) (Katsuyama, 2005).
This paper focusses on the GS15923 line, because well organized wings with antero-posterior and dorso-ventral axes were induced in the eye field when GS15923 was crossed with ey-GAL4. Using the inverse PCR method, the insertion site of the GS vector in GS15923 was determined and a predicted gene, CG31151, was found adjacent to the insertion site. CG31151 was the only gene expressed in a GAL4-dependent manner in GS15923. After cloning cDNAs corresponding to CG31151, an ORF of a previously uncharacterized gene was identified that was named winged eye (wge), which has a 345-bp extension at the 5' end from the estimated translation initiation site of expressed sequence tag (EST) clones corresponding to CG31151. The sequence of an EST clone, LP24488, overlapped by 693 bp with the 5' end of the cloned cDNA and 20 bp of the 5' end of the RA transcript, confirming the identified ORF. wge encodes a 1,658-aa protein with two Gln-rich, one Ala-rich, and one Ser-rich domain at the N-terminal half and a bipartite nuclear localization signal and a BAH domain in the C-terminal half, implying that Wge is involved in epigenetic regulation of gene expression. This prediction is consistent with the specific localization of Wge on polytene chromosomes. Ectopic wing induction by forced wge expression was confirmed by using UAS-wge transgenic flies. The Wge-mediated ectopic wings had the costa with spine bristles, the triple row of bristles, and the double row of bristles that are formed at the anterior-proximal part of the wing, at the anterior wing margin, and at the distal wing margin in wild-type wing, respectively, indicating that Wge-mediated ectopic wings are correctly organized along the antero-posterior and dorso-ventral axes. In contrast, Vg-mediated ectopic wings in the eye field are outgrowths with wing hair that do not have any wing margins, suggesting that Wge is involved in the wing formation upstream of Vg (Katsuyama, 2005).
Database analysis revealed a genome sequence of an Anopheles gambiae gene with striking similarity to wge; for example, there was a 77% amino acid identity in the BAH domain. Comparison of the two sequences led to the identification of a previously uncharacterized protein domain named highly corresponding region (HCR: 126 aa, amino acids 1130–1255) with similarity with the A. gambiae gene product (57% amino acid identity), KIAA1447 human protein (41%), CAGL79 human protein (33%), and BC060615 mouse protein (42%). These results suggest that wge is evolutionarily conserved in insects and mammals (Katsuyama, 2005).
It has been demonstrated that, in combination with activation of Notch signaling, expression of Antp by ey-GAL4 induces ectopic wings with antero-posterior and dorso-ventral axes, that are similar to Wge-mediated ectopic wings. To investigate the requirement of Antp for the Wge-mediated induction of ectopic wings, transgenic flies were generated possessing an inverted repeat (IR) expression construct of Antp cDNA that specifically inhibits Antp expression in a GAL4-dependent manner due to RNA interference. Antp-IR expression inhibited the formation of ectopic wings induced by the expression of Antp and Notch signaling activation, indicating that the construct works as a specific inhibitor of Antp expression. Antp-IR expression, however, does not inhibit the formation of ectopic wings when coexpressed with wge. Consistent with this result, forced expression of wge does not induce ectopic expression of Antp in eye discs of ey-GAL4;UAS-wge larvae. These results indicated that wge overexpression induces ectopic wings in an Antp-independent manner. Moreover, Wge-IR expression suppresses the formation of ectopic wings induced by the expression of Antp and Notch signaling activation. These results indicate that Wge acts downstream of Antp and Notch signaling in the ectopic wing induction (Katsuyama, 2005).
Ectopic expression of vg in various imaginal discs induces ectopic wing-like outgrowth. In combination with Wg signaling, however, it induces wings with wing margins. Wg signaling is also suggested to participate in the determination of wing discs. Whether wge overexpression induces ectopic expression of Vg and Wg was investigated in eye imaginal discs when it induces ectopic wings with wing margins. In wild type, Vg is expressed in the wing discs but not in eye discs, whereas, in ey-GAL4;UAS-wge, there was significant expression of Vg in the eye discs. The forced expression of wge also activates vg D/V boundary enhancer-lacZ (vgBE-lacZ). These results indicate that wge overexpression is sufficient to induce the ectopic expression of vg, a control gene for wing formation. In third-instar larvae, Wg was expressed at the peripheral edge of the dorsal and ventral sides of wild-type eye discs. Overexpression of wge by ey-GAL4 enhances the dorsal expression of Wg and suppresses the ventral expression of Wg in eye discs. These results are consistent with the findings that wge overexpression induces ectopic wings at the dorsal part of the eye field. Therefore, Wge is suggested to induce ectopic wings upstream of Vg and Wg (Katsuyama, 2005).
The effects of wge overexpression on the expression of genes involved in the determination of eye identity, such as ey and eya, was investigated. Overexpression of wge was seen to repress Eya expression at the dorsal side of the eye discs, in contrast to Wg expression, which is up-regulated at the dorsal side by Wge. Similar results were obtained with eya-lacZ transgenic larvae. The enhancer trap line of eya-lacZ reflects endogenous expression of Eya. Double staining revealed that the ectopic induction of Vg does not overlap with Eya expression when wge was overexpressed in eye discs. These results suggest that Wge-mediated down-regulation of eya is involved in the ectopic induction of vg. However, it was not possible to examine the effects of eya overexpression on Wge-mediated ectopic vg expression, because eya overexpression in eye discs inhibits eye disc development. Further analysis is required to determine the relationship between the down-regulation of eya and the ectopic induction of vg (Katsuyama, 2005).
Clonal activation of Wg signaling represses eya expression in eye discs. To examine the effects of clonal induction of wge overexpression on eya and wg expression, a cell lineage tracer technique was applied by using a combination of the flp/FRT and GAL4/UAS recombinase systems. In this system, in which wge-overexpressing cells are labeled with GFP, eya-lacZ expression is repressed by clonal induction of wge overexpression in a cell-autonomous manner, whereas Wg expression is not induced when the wge-expressing clone is induced, even on the dorsal side of the eye discs. These results indicate that overexpression of wge represses expression of eya in eye discs in a cell-autonomous manner, independent of the up-regulation of wg. Overexpression of wge-inhibits expression of eya, a gene downstream of ey; however, ey-lacZ expression in eye discs is not repressed by clonal induction of wge overexpression. Consistent with these results, Ey-mediated ectopic induction of eya is inhibited by coexpression of wge. Ectopic induction of ey under the control of dpp-GAL4 induces ectopic induction of eya-lacZ in the leg, wing, and antennal discs, but the ectopic expression of eya-lacZ is totally suppressed by the coexpression of wge. Coexpression of GFP does not suppress the Ey-mediated induction of eya, indicating a specific effect of wge overexpression on eya expression. These results suggest that overexpression of wge suppresses the eye development program downstream of ey. Eya repression occurs only on the dorsal side of the eye disc when wge is overexpressed with ey-GAL4, although clonally overexpressed wge also represses Eya on the ventral side. Further analysis is required to explain these phenomena (Katsuyama, 2005).
Although ey-GAL4-dependent overexpression of wge-induced Wg expression on the dorsal side of the eye discs, clonal induction of wge overexpression did not induce Wg expression in eye discs. Consistent with these results, clonal induction of wge overexpression did not induce either ectopic expression of Vg in eye discs or ectopic formation of wings in the eye field on the head, suggesting that overexpression of wge in a relatively large field is required for the transformation of eye to wing. Repression of eya but absence of vg expression in clonal overexpression of wge might represent an intermediate step toward wing transformation, which has to be analyzed further. In wing discs, clonal induction of wge overexpression did not affect expression of either Vg or Wg, indicating a specific effect of clonal induction of wge overexpression on eya expression in eye discs (Katsuyama, 2005).
To investigate the requirement of wge for wing development, a wge-deficient mutant was generated by mobilizing the P element. The deletion of wge was screened by using genomic PCR in 126 excision lines. In one line, Wge40, sequencing analysis after genomic PCR revealed that a 2,215-bp sequence, including the wge first exon, was deleted. In Wge40, there was no wge expression, and the expression of a gene neighboring Wge, Irp-1A, was not affected, indicating that Wge40 is a wge null mutant. The embryogenesis of Wge40 was quite normal, but the development of Wge40 gradually stopped after the first-instar larval stage, suggesting crucial roles of wge in larval development or growth. A rare rescue (3%) of larval lethality was observed by wge overexpression by using a heat-shock promoter, reflecting the context-dependent requirement of wge for development. Rescue was never observed, however, in the absence of heat shock (29°C for 2 h every 24 h). wge mutant clones were then induced by using the flp/FRT system with the Minute technique. When the wge mutant clones were introduced in wing discs 48 h AED by heat shock, no Vg expression was observed in many cells within the clones, but some cells in the clones still expressed Vg. The expression of dpp-lacZ was observed in the wge mutant clones, indicating a specific effect of the wge mutation on vg expression. The size of these clones is relatively small, whereas Vg expression is not affected in all but a few small-sized clones when the wge mutant clones are introduced 72 h AED. These results indicate that wge is required for vg induction in wing discs in a context-dependent manner. Compared with the wge mutant clones that were introduced 72 h AED, the wge mutant clones that were introduced 48 h AED were small in size, suggesting stage-specific involvement of wge on the growth of disc cells (Katsuyama, 2005).
Clonal induction of wge overexpression represses eya-lacZ expression in eye discs. Derepression of eya was investigated in the wge mutant clones. Contrary to prediction, there was no misexpression of Eya in wge mutant clones that were introduced at both 48 and 72 h AED in imaginal discs such as antennal, leg, and wing discs. In eye discs, Eya was not expressed in many cells within the wge mutant clones but was expressed in some cells when the clones were introduced 48 h AED, whereas the expression of Eya was not affected in the clones when the wge mutant clones were introduced 72 h AED. These results indicate that wge is required for eya expression in eye discs in a context-dependent manner, which is similar to the function of wge in the regulation of vg expression in wing discs. Consistent with the requirement of wge for the function of vg and eya, adult structures, such as eyes, wings, and legs, were malformed when wge mutant clones were introduced 72 h AED. The mutant clones were distinguished by the absence of Ubi-GFP. Pupal lethality was induced when wge mutant clones were introduced 48 h AED. The participation of wge in the development and growth of various appendages confirmed the ubiquitous expression of wge. RT-PCR findings revealed constitutive expression of wge throughout the larval and pupal developmental stages and ubiquitous expression of wge in larval tissues and imaginal discs. The ubiquitous expression of wge in various tissues was confirmed by in situ hybridization experiments (Katsuyama, 2005).
Wge has a BAH domain that is frequently found in proteins participating in the epigenetic regulation of gene expression. To investigate the nuclear localization and chromatin association of Wge, FLAG-tagged wild-type Wge and FLAG-tagged mutant protein (deltaBAH) lacking the BAH domain was expressed and salivary glands were stained with anti-FLAG antibody. Both wild-type Wge and DeltaBAH were localized in the nuclei of the salivary glands, whereas only wild-type protein, and not DeltaBAH, localized at specific sites on polytene chromosomes. These results indicate that Wge associates with chromatin in a BAH domain-dependent manner. The association of Wge with chromatin seems to be crucial for Wge function, because DeltaBAH does not induce ectopic wings in the eye field when it is expressed by ey-GAL4 (Katsuyama, 2005).
The binding sites of Wge were analyzed with DAPI staining (DNA) under higher magnification. Some signals overlapped with DAPI staining and others did not overlap with DAPI staining, suggesting that Wge localizes in both bands and interbands of polytene chromosomes. The Wge binding sites on polytene chromosomes were compared to that of Posterior sex combs (PSC). Almost all PSC binding sites were coincident with some Wge binding sites, suggesting that some Wge function is related to PSC function. The genetic interaction between wge and Psc was investigated with Wge40 and Psc1. The extra sex comb phenotype of Psc1 was suppressed by the loss of one dose of wge, suggesting a wge function similar to that of trithorax-group (trxG) genes, which antagonize PcG genes. There was a maternal effect in Wge40 single heterozygotes, as indicated by the appearance of an additional sex comb on the second tarsomere of the first leg. Such a transformation of the second to the first tarsomere of the leg occurs in some PcG mutants such as multi sex combs and cramped. The additional sex comb phenotype of Wge40 was suppressed by a partial loss-of-function of Psc. In both Wge40 and Psc1 single heterozygotes, the number of sex comb teeth on the first tarsomere of the first legs was increased, whereas there was no significant modification of the number of sex comb teeth in double heterozygotes. These results indicate that wge and Psc have antagonistic roles in both transformation from the second thoracic legs to the first thoracic legs and transformation from the second tarsomere to the first tarsomere of the first leg but not in the increase in the number of sex comb teeth (Katsuyama, 2005).
Thus, Wge localizes at specific sites on polytene chromosomes in a BAH domain-dependent manner, and wge overexpression induces a gain-of-function transformation of eyes to wings. The extra sex comb phenotype of Psc1 is suppressed by the loss of one dose of wge. The characteristics of wge are similar to that of trxG genes. The trxG genes act as suppressors of the Polycomb phenotype and are implicated in the activation of Hox selector genes. Therefore, similar to loss-of-function mutations in the PcG genes, gain-of-function mutations in trxG genes cause ectopic expression of Hox selector genes and homeotic transformations. TrxG proteins also localize at specific sites on polytene chromosomes, and one of the trxG proteins, ASH1, has a BAH domain. There are several functional differences, however, between wge and trxG genes. One major functional difference is Hox selector gene independence on homeotic transformation. Mutations of the trxG genes cause homeotic transformations through the modulation of transcriptional regulation of the Hox selector genes. In contrast, overexpression of wge induces ectopic wings in an Antp-independent manner. Although endogenous wing development is considered to be independent of Antp, Antp is the only Hox selector gene examined that induced eye-to-wing transformation in the system. In addition, wge overexpression does not induce ectopic expression of Antp in eye discs. Therefore, wge induces eye-to-wing transformation in an independent Hox selector gene. Moreover, wge is required for the ectopic wing formation that is induced by the expression of Antp and the activation of Notch signaling. These results suggest that wge is involved in the regulation of field-specific selector gene expression but not in the regulation of region-specific Hox selector gene expression. There is probably a regulatory mechanism that determines the field-specific identity after determination of region-specific identity by Hox selector genes. Another difference between wge and trxG is that the trxG functions as activators, whereas wge overexpression also represses eya (Katsuyama, 2005).
wge is required for the expression of both vg in wing discs and eya in eye discs in a context-dependent manner. Overexpression of wge, however, induces ectopic expression of vg and represses eya expression in eye discs. In wing discs, wge overexpression does not induce either ectopic expression of vg or repression of vg. Therefore, wge regulates expression of vg and eya in a context-dependent manner. Consistent with the context-dependent function of wge, wge is expressed ubiquitously throughout larval to pupal development and in various tissues. The field-specific identity should be determined from an equivalent group of cells. This characteristic is observed not only in normal development but also in artificial situations of imaginal discs called transdetermination, in which, after regenerative cell growth, disc cells change their determined state to another determined state, e.g., a leg disc transdetermines to a wing disc. Transdetermination is a polyclonal event and not the result of either differentiation of reserve cells or somatic mutations. Context-dependent regulation of gene expression by a ubiquitously expressed gene might explain how differences are created within a group of equivalent cells (Katsuyama, 2005).
The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, a combinatorial strategy was used to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. To overcome the lack of high-quality consensus binding site sequences, phylogenetic shadowing of known Ey binding sites in sine oculis (so) was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yielded a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach to identify direct downstream targets for any transcription factor genome-wide (Ostrin, 2006).
The extensive genetic regulatory flows underlying specification of different neuronal subtypes are not well understood at the molecular level. The Nplp1 neuropeptide neurons in the developing Drosophila nerve cord belong to two sub-classes; Tv1 and dAp neurons, generated by two distinct progenitors. Nplp1 neurons are specified by spatial cues; the Hox homeotic network and GATA factor grn, and temporal cues; the hb -> Kr -> Pdm -> cas -> grh temporal cascade. These spatio-temporal cues combine into two distinct codes; one for Tv1 and one for dAp neurons that activate a common terminal selector feedforward cascade of col -> ap/eya -> dimm -> Nplp1. This study molecularly decodes the specification of Nplp1 neurons, and finds that the cis-regulatory organization of col functions as an integratory node for the different spatio-temporal combinatorial codes. These findings may provide a logical framework for addressing spatio-temporal control of neuronal sub-type specification in other systems (Stratmann, 2017).
The Drosophila ventral nerve cord (VNC; defined here as thoracic segments T1-T3 and abdominal A1-A10) contains ~10,000 cells at the end of embryogenesis, which are generated by a defined set of ~800 neuroblasts (NBs). The Apterous neurons constitute a small sub-group of interneurons, identifiable by the selective expression of the Apterous (Ap) LIM-homeodomain factor, as well as the Eyes absent (Eya) transcriptional co-factor and nuclear phosphatase. A subset of Ap neurons express the Nplp1 neuropeptide, but can be sub-divided into the lateral thoracic Tv1 neurons, part of the thoracic Ap cluster of four cells, and the dorsal medial row of dAp neurons. In line with the distinct location of the Tv1 and dAp neurons, studies have revealed that they are generated by distinct NBs; NB5-6T and NB4-3, respectively. A number of studies have addressed the genetic mechanisms underlying the specification of the Tv1 and dAp neurons, and the regulation of the Nplp1 neuropeptide. These have revealed that two distinct spatio-temporal combinatorial transcription factor codes, one acting in NB5-6T and the other in NB4-3, converge on a common initiator terminal selector gene; collier, encoding a COE/EBF transcription factor. Col in turn is necessary and sufficient to trigger a feed forward loop (FFL) consisting of Ap, Eya and the Dimmed (Dimm) bHLH transcription factor, which ultimately activates the Nplp1 gene. Strikingly, the combinatorial coding selectivity of the spatio-temporal cues combined with the information-coding capacity of the FFL results in the selective activation of Nplp1 in only 28 out of the ~10,000 cells within the VNC. While these genetic studies have helped resolve the regulatory logic of this cell specification event, they have not addressed the molecular mechanisms by which the two different spatio-temporal combinatorial codes intersect upon the col initiator terminal selector, to trigger a common terminal FFL, or the molecular nature of the FFL (Stratmann, 2017).
To address this issue, this study has identified enhancers for Tv and dAp neuron expression for the genes in the common Tv1/dAp FFL: col, ap, eya, dimm and Nplp1. Transgenic reporters were generated for these enhancers, both wildtype and mutant for specific transcription factor binding sites, to test their regulation in mutant and misexpression backgrounds. CRISPR/Cas9 technology was used to delete these enhancers in their normal genomic location to test their necessity for gene regulation. Strikingly, this study found that the distinct upstream spatio-temporal combinatorial codes, which trigger col expression in Tv1 versus dAp neurons, converge onto different enhancer elements in the col gene. Hence, the col Tv1 neuron enhancer is triggered by Antp, hth, exd, lbe and cas, while the dAp enhancer is triggered by Kr, pdm and grn. In contrast to this subset-specific enhancer set-up for col activation, the subsequent, col-driven Nplp1 FFL feeds onto common enhancers in each downstream gene. These findings reveal that distinct spatio-temporal cues, acting in different neural progenitors, can trigger the same FFL by converging on discrete enhancer elements in an initiator terminal selector, to thereby dictate the same ultimate neuronal subtype cell fate (Stratmann, 2017).
This study has been able to molecularly decode the Tv1/dAp genetic FFL cascades, bolstering evidence for a complex molecular FFL, based upon sequential transcription factor binding to the downstream genes. The NB4-3 and NB5-6T neuroblasts are born in different regions of the VNC, and express different spatial determinants i.e., Antp, Lbe, Hth, Exd and Gr. As lineage progression commences, they undergo a programmed cascade of transcription factor expression; the temporal cascade. Early temporal factors Kr and Pdm integrate with Grn in NB4-3, while the late temporal factor Cas integrates with Antp, Lbe, Hth and Exd in NB5-6T, to create two distinct combinatorial spatio-temporal codes. These two codes converge on two different enhancers in the col gene, triggering Col expression, and hence the Nplp1 FFL. The FFL, in this case a so-called coherent FFL, where regulators act positively at one or several steps of a cascade, was first identified in E.coli and yeast regulatory networks, but have also been identified in C.elegans and Drosophila. Coherent FFLs can act as regulatory timing devices, exemplified by the action of col in NB5-6T: The initial expression of col in Ap cluster cells triggers a generic Ap/Eya interneuron fate in all four cells, while its downregulation in Tv2-4 and maintenance in Tv1 helps propagate the FFL leading to Nplp1 expression (Stratmann, 2017).
This study has found that the two different spatio-temporal programs converge on col, but on different enhancer elements. However, neither enhancer element gave complete null effects when deleted. Specifically, the 6.3kb col-Tv-CRM shows robust reporter expression, overlaps with endogenous col expression, responds to the upstream mutants, and is affected by TFBS mutations. However, when deleted (generating the colΔTv-CRM mutant), it had weak effects upon endogenous col expression in NB5-6T, and no effect upon Eya and Nplp1 expression. Deletion of the col-dAp-CRM (generating the colΔdAp-CRM mutant), gave more robust effects with reduction of Col, Eya and Nplp1 in dAp cells, although the expression was not lost completely (Stratmann, 2017).
Early developmental genes, which often are dynamically expressed, may be controlled by multiple enhancer modules, to thereby ensure robust onset of gene expression. This has been reported previously in studies of early mesodermal and neuro-ectodermal development, in which several genes i.e., twist, sog, snail are controlled by multiple distal enhancer fragments, so called 'shadow enhancers', in order to ensure reliable onset of gene expression. The shadow enhancer principle is also supported by recent findings on the Kr gene. Moreover, extensive CRM transgenic analysis, scoring thousands of fragments in transgenic flies, has also supported the shadow enhancer idea, revealing that a number of early regulators, several of which encode for transcription factors, indeed have shadow enhancers. The dichotomy between the col transgenic reporter results and the partial impact on col expression upon deletion of its Tv1 and dAp enhancers, gives reason to speculate that col may be under control of additional enhancers, some of which may be referred to as shadow enhancers (Stratmann, 2017).
The results on the eya, ap, dimm and Nplp1 enhancer mutants stand in stark contrast to the col CRMs findings. For these four genes, the enhancer deletion resulted in robust, near null effects, on expression. It is tempting to speculate that the current findings, combined with previous studies, points to a different logic for early regulators, with highly dynamic patterns, requiring several functionally overlapping enhancers for fidelity, and late regulators and terminal differentiation genes, which may operate with one enhancer that is inactive until the pertinent combinatorial TF codes have been established (Stratmann, 2017).
Analysis of the ap and eya enhancers indicates that Col directly interacts with these enhancers. Both of these enhancer-reporter transgenes are affected in col mutants, and can be activated by ectopic col. Moreover, mutation of one Col binding site in the ap enhancer and two sites in the eya enhancer, was enough to dramatically reduce enhancer activity. Direct action of Col on ap and eya is furthermore supported by recent data on Col genome-wide binding, using ChIP, which demonstrated direct binding of Col to these regions of ap and eya in the embryo. The regulation of ap is an excellent example of the complexity of gene regulation, and studies have identified additional enhancers controlling ap expression in the wing, muscle and brain (Stratmann, 2017).
In contrast to regulation of ap and eya, a direct action of Col on dimm and Nplp1 is less clear. Analysis of the dimm and Nplp1 enhancers did not reveal perfectly conserved Col binding sites. Mutation of multiple non-perfect Col binding sites in the dimm enhancer did not affect reporter expression in the Ap cluster, but did however reduce levels in the dorsal Ap cells. Mutation of non-perfect Col binding sites in the Nplp1 enhancer had no impact on enhancer activity, neither in Tv1 nor dAp. These findings support a model where Col is crucial for directly activating ap and eya, which in turn directly activate dimm and Nplp1, with some involvement of Col on dimm. However, support for a direct role for Col on Nplp1 comes from RNAi studies in larvae or adult flies, showing that knockdown of col resulted in loss of Nplp1, while Ap, Eya and Dimm expression was unaffected (Stratmann, 2017).
It is tempting to speculate that Col regulates Nplp1 not via direct interaction with its enhancer, but rather as a chromatin state modulator, keeping the chromatin around the Nplp1 locus in an accessible state, in order for Dimm, Ap and Eya to be able to access the Nplp1 gene. Support for this notion comes from studies on the mammalian Col orthologue EBF, which is connected to the chromatin remodeling complex SWI/SNF during EBF-mediated gene regulation in lymphocytes (Gao, 2009). Moreover, the central SWI/SNF component Brahma was recently identified in a genetic screen for Ap cluster neurons, and found to affect FMRFa neuropeptide expression in Tv4 without affecting Eya expression, indicating a late role in Ap cluster differentiation. Alternatively, Col may activate Nplp1 via unidentified, low affinity sites, similar to the mechanism by which Ubx regulates some of its embryonic target genes (Stratmann, 2017).
ap encodes a LIM-HD protein, a family of transcription factors well known to control multiple aspects of terminal neuronal subtype fate, including neurotransmitter identity, axon pathfinding and ion channel expression. The current results indicate that Ap in turn acts upon dimm, and subsequently with Dimm on Nplp1. eya encodes an evolutionary well-conserved phosphatase and does not bind DNA directly, instead acting as a transcriptional co-factor. Eya (and its orthologues) have been found to interact with several transcription factors in different systems, but whether it forms complexes with Col and Ap is not known (Stratmann, 2017).
The final transcription factor in the FFL is Dimm, a bHLH protein. Dimm is selectively expressed by the majority of neuropeptide neurons in Drosophila, and is important for expression of many neuropeptides. Intriguingly, Dimm is also both necessary and sufficient to establish the dense-core secretory machinery, found in neuropeptide neurons. Based upon these findings Dimm has been viewed as a cell type selector gene, acting to up-regulate the secretory machinery. This study found evidence for that Dimm acts directly on the Nplp1 enhancer, and this raises the possibility that Dimm is both a selector gene for the dense-core secretory machinery, and can act in some neuropeptide neurons to directly regulate specific neuropeptide gene expression (Stratmann, 2017).
Eyes absent (Eya) is a highly conserved transcriptional coactivator and protein phosphatase that plays vital roles in multiple developmental processes from Drosophila to humans. Eya proteins contain a PST (Proline-Serine-Threonine)-rich transactivation domain, a threonine phosphatase motif (TPM), and a tyrosine protein phosphatase domain. Using a genomic rescue system, this study finds that the PST domain is essential for Eya activity and Dac expression, and the TPM is required for full Eya function. The threonine phosphatase activity plays only a minor role during Drosophila eye development and the primary function of the PST and TPM domains is transactivation that can be largely substituted by the heterologous activation domain VP16. A primary function of Eya during Drosophila eye development is as a transcriptional coactivator. Moreover, the PST/TPM and the threonine phosphatase activity are not required for in vitro interaction between retinal determination factors. Finally, this work is the first report of an Eya-Ey physical interaction. These findings are particularly important because they highlight the need for an in vivo approach that accurately dissects protein function (Jin, 2016).
The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. This study explores the mechanisms by which eya expression is spatially and temporally governed in the developing eye. Multiple cis-regulatory elements function cooperatively to control eya transcription and spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, it was shown that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis (Weasner, 2016).
The transition from proliferation to specification is fundamental to the development of appropriately patterned tissues. In the developing Drosophila eye, Eyes absent (Eya) and Sine oculis (So) orchestrate the progression of progenitor cells from asynchronous cell division to G1 arrest and neuronal specification at the morphogenetic furrow. This study uncovered a novel role for Eya and So in promoting cell cycle exit in the Second Mitotic Wave (SMW), a synchronized, terminal cell division that occurs several hours after passage of the furrow. Combgap (Cg), a zinc-finger transcription factor, antagonizes Eya-So function in the SMW. Based on Cg's ability to attenuate Eya-So transcriptional output in vivo and in cultured cells and on meta-analysis of their chromatin occupancy profiles, it is speculated that Cg limits Eya-So activation of select target genes posterior to the furrow to ensure properly timed mitotic exit. This work supports a model in which context-specific modulation of transcriptional activity enables Eya and So to promote both entry into and exit from the cell cycle in a distinct spatiotemporal sequence (Davis, 2017).
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