sine oculis
Drosophila eye development is under the control of early eye specifying genes including eyeless (ey), twin of eyeless (toy), eyes absent (eya), dachshund (dac) and sine oculis (so). They are all conserved between vertebrates and insects and they interact in a combinatorial and hierarchical network to regulate each other's expression. so has been shown to
be directly regulated by ey through an eye-specific enhancer (so10). The regulation of this element has been studied; both Drosophila Pax6 proteins, namely Ey and Toy, bind and positively regulate so10 expression
through different binding sites. By targeted mutagenesis experiments, these Ey and Toy binding sites were disrupted and their functional
involvement in the so10 enhancer expression in the eye progenitor cells was studied. A differential requirement has been shown for the Ey and Toy binding sites in
activating so10 during the different stages of eye development. Additionally, in a rescue experiment performed in the so1 mutant, the Ey
and Toy binding sites were shown to be required for compound eye and ocellus development, respectively. Altogether, these results suggest a differential
requirement for Ey and Toy to specify the development of the two types of adult visual systems, namely the compound eye and the ocellus (Punzo, 2002).
All animals analyzed so far, ranging from flatworms to mammals, have a Pax6 gene that is universally required for eye specification, according to the current state of knowledge. In contrast to vertebrates, where generally a single Pax6 gene gives rise to several differentially spliced transcripts, Drosophila and other holometabolous insects have two Pax6 genes, raising the question of functional redundancy. Gene duplication and subsequent divergence of developmental control genes is a major driving force in evolution, increasing the diversity and complexity of the organisms. A second mechanism for recruiting additional genes into a developmental pathway is enhancer fusion. The acquisition of new cis-regulatory elements represents an important mechanism for functional diversification. The findings reported in this study strongly support both of these hypotheses, since toy is able to rescue the eye development in an ey mutant when expressed in the ey domain. The finding that ey and toy exhibit different expression patterns during embryogenesis might account in part for their functional biological diversity. In the eye, both genes are co-expressed, except for the ocellar territory where only toy is expressed. In addition, it has been proposed that Toy and Ey diverged to regulate different sets of target genes because of a N14G mutation that changes the DNA binding specificity of the PD domain of ey. Indeed, using the so10 regulatory element it was found that Toy does not bind to the same sequences as Ey, but interestingly, Toy and Ey regulate the same target enhancer in different cells. The phenotypes obtained in rescue experiments using either the Ey/Toy or Toy binding site mutated enhancers, nicely parallel the phenotypes observed in ey mutants. The ey null mutant still has ocelli but lacks compound eyes. Interestingly escapers from the recently isolated toy mutant (toyG7.39) exhibit no eye reduction whereas the ocelli are partially missing. Therefore, removal of the common target gene of both Pax6 proteins in the eye (e.g. so1 mutant) consequently leads to a loss of both compound eyes and ocelli. Therefore, it is proposed that one of the developmental programs of toy is in part to specify ocellar development in addition to head formation, since toy mutants generated are characterized by pupal lethality, pharate adults lacking half of the head or the entire head capsule. Thus, it is proposed that the so gene is regulated by toy to specify the ocelli and by ey to specify the compound eyes during larval development (Punzo, 2002).
The analysis of ey and toy allows for the dissection of the evolutionary changes after the gene duplication event that happened during insect evolution. (1) The cis-regulatory regions of the two genes have diverged, leading to both temporal and spatial changes of expression; toy is expressed much earlier than ey during embryogenesis, whereas ey is not expressed in the ocellar region of the larval eye disc. (2) The protein coding regions of the two genes have diverged, most importantly in the paired domain where asparagine 14 (which is present in most Pax6 homologs), has been mutated in ey to glycine, which changes the DNA binding properties of the protein significantly. (3) The positive autocatalytic feedback loop found in vertebrates for their single Pax6 gene, has evolved into a heterocatalytic control loop in which toy transcriptionally activates ey by binding to the eye-specific enhancer of ey. (4) Both toy and ey cooperate in differentially regulating the so target gene, reflecting the fact that earlier in evolution so was regulated by a single Pax6 gene. These findings strongly support the hypothesis of intercalary evolution showing that the ey gene has been intercalated into the eye developmental pathway between toy and so. The observation that toy activates ey in the eye progenitor cells of the embryo, where neither so and eya are expressed, indicates that toy and ey are acting high up in the genetic hierarchy leading to eye development (Punzo, 2002).
In Drosophila, the sine oculis (so) gene is
important for the development of the entire visual system, including Bolwig's
organ, compound eyes and ocelli. Together with twin of eyeless,
eyeless, eyes absent and dachshund, so
belongs to a network of genes that by complex interactions initiate eye
development. Although much is known about the genetic interactions of the
genes belonging to this retinal determination network, only a few such
regulatory interactions have been analysed down to the level of DNA-protein
interactions. An eye/ocellus
specific enhancer of the sine oculis gene has been identified that is directly regulated
by eyeless and twin of eyeless. This regulatory element has been further characterized
and a minimal enhancer fragment of
so has been identified that sets up an autoregulatory feedback loop crucial for proper
ocelli development. By systematic analysis of the DNA-binding specificity of
so the most important nucleotides for this interaction have been identified.
Using the emerging consensus sequence for SO-DNA binding a
genome-wide search was performed and eyeless has been identified as
well as the signalling gene hedgehog as putative targets of
so. These results strengthen the general assumption that feedback loops
among the genes of the retinal determination network are crucial for proper
development of eyes and ocelli (Pauli, 2005).
In-vitro data on the autoregulatory element with the known so
target sequence of lz and the AREC3/Six4-binding site, the consensus
sequence GTAANYNGANAYC/G was identified as necessary for SO binding to DNA. This consensus
sequence was taken as a basis for scanning the Drosophila genome for similar
sites. In total, 1632 putative so targets emerged
from this survey. Out of the affected genes several candidates are already known
to be involved in eye development (Pauli, 2005).
so gene activity is crucial for proper development of the entire
visual system of Drosophila, including the larval visual
system (Bolwig's organ), the optic lobe, the compound eye and the ocellus.
An eye-specific enhancer of
so, so10, has been identified that is regulated by ey and toy. When used
as a driver for so, so10 is sufficient to rescue only eye development
of so1 mutant flies but not ocellus development. A fragment of
27 bp, soAE, found downstream of so10, is sufficient
to rescue the entire mutant phenotype of so1 mutant flies
when combined with so10. The So protein itself binds to soAE and,
in cooperation with Eya, forms an autoregulatory feedback loop that is
essential for ocellus development (Pauli, 2005).
Since So binds to its own enhancer and autoregulation cannot initiate
expression of a gene, the initiation of so expression in the ocellar
region must be triggered by other means. The following model is proposed.
Initiation of so expression in early third instar eye discs is
mediated by ey and toy throughout the eye disc, including
the ocellar precursors. Later, after this first induction, so
cooperatively with eya can maintain its own expression in the ocellar
region by a positive autoregulatory feedback. Thus, the initiation of
so expression is mediated by so10, whereas for the maintenance of
so, soAE is required. This is supported by the observation
that so10, which is activated by ey and toy, mediates
expression in early third instar larvae all over the eye disc and only later
gets restricted to the compound eye part (Pauli, 2005).
In this model the specificity of so expression for ocellar
precursor cells is provided by the expression pattern of eya; Eya
protein can be found only in the ocellar region itself, where it specifically
interacts with So, and no Eya is present in the proximity of these cells. The
importance of eya is further strengthened by the fact that
eya4 mutants show an eyeless and ocelliless phenotype.
Therefore, to elucidate the mechanisms that control gene expression
specifically in ocellar precursor cells, additional studies on eya are required (Pauli, 2005).
Positioned at the top of the hierarchy of the retinal determination
network, ey is a potent inducer of ectopic eyes and is able to
directly induce so and eya. Like ey, so and
eya are able to induce ectopic eyes but only when co-expressed;
so alone fails to do so (Pauli, 2005).
To accomplish this induction, eya and so need to feed
back on ey, obviously by binding to the eye-specific enhancer of
ey. In an ectopic situation, the feedback of so/eya on
ey is strong enough to induce ey for ectopic eye
formation (Pauli, 2005).
The function of this feedback loop in normal eye development remains to be
elucidated. so and eya are both expressed posterior to the
furrow and are important for neuronal development.
Nevertheless, ey is tuned down posterior to the MF. The activity of
the so-binding site in the ey gene might, therefore, be
suppressed by other factors or by so itself during cellular
differentiation posterior to the furrow. Since co-expression of ey, so
and eya is elevated only in a few cells in front of the MF and within
the MF, a possible role for this feedback loop might be to boost ey
expression in front of and within the furrow, which leads to a strengthening
of so and eya expression in just a few cell rows (Pauli, 2005).
For proper eye development, a well-balanced expression level of the genes
belonging to the retinal determination network is crucial. Loss-of-function
mutations, as well as overexpression of the eye specification genes ey,
eya, so or dac during eye development, impede proper
determination of the organ and result in a reduction in eye size.
Therefore, it is hypothesized that a feedback loop of so on ey
is also important for the fine-tuning of ey expression during normal
eye development. Due to its previously proposed ability to activate as well as
to repress the expression of genes, so is a potent regulator in this
context (Pauli, 2005).
decapentaplegic (dpp) signalling plays an important role
in the complex regulatory network of eye development. In dpp mutant
eye discs, so, eya and dac are not expressed, whereas
dpp is able to initiate ectopic expression of so and
dac when expressed at the anterior margin of the eye disc.
Conversely, dpp expression is patchy in eye discs of eya and
so loss-of-function mutants, suggesting that eya and
so are required for either initiation or maintenance of dpp
at the posterior disc margin before MF initiation (Pauli, 2005).
hh is required for dpp expression at the posterior margin
before MF initiation, and dpp expression is induced by hh in
the MF, supporting the assumption that dpp is downstream
of hh signalling. Since dpp alone is not able to rescue
posterior margin clones of hh, there have to be more eye-relevant
target genes of hh signalling during third instar larval development.
dpp in combination with eya can restore photoreceptor
differentiation in posterior margin clones lacking smoothened
(smo) expression (smo is a cell-autonomous receptor of
hh signalling). This shows that dpp, in combination with
eya, is able to bypass the requirement of hh during eye
development. Taken together, it is evident that hh is necessary
for proper eya and dpp expression, both of which can induce
so, and it contains two so target sites. It is therefore
hypothesized that the transcriptional complex consisting of Eya and So, as
with ey, might also feed back on hh in order to drive the
furrow during late eye development. In this model the genetic cascade starts
with hh, which induces dpp and eya, moves on to
so and through the So/Eya complex feeds back to hh in order
to maintain hh expression as a driving force of the MF (Pauli, 2005).
The impact of these so-binding sites in the hh enhancer
on eye development becomes evident from the fact that hh1
(bar-3) mutant flies have smaller eyes. The severity of the
hh1 mutant phenotype is probably diminished by an
additional putative So-binding site that resides outside the area covered by
the hh1 deletion. If functional, this region (5' to
the hh1 deletion) might mediate a residual
hh-expression that overcomes the loss of the other sites to some
extent. Another possible explanation for the rather weak
hh1 phenotype might be that the feedback of so on
hh is not crucial for MF initiation but still might be of importance
for the well-balanced expression of hh during MF propagation (Pauli, 2005).
so belongs to the Six gene family. All Six proteins are
characterized by a Six domain and a Six-type homeodomain, both of which are
essential for specific DNA binding and protein-protein interaction. Based on
the amino acid sequence of their homeodomain and Six domain, the Six genes
were divided into three subgroups. Each of the three Drosophila
homologues can be assigned to one of these subgroups: so is mostly
related to Six1/2, optix to Six3/6 and DSix4 to
Six4/5 (Pauli, 2005).
Promoter analyses of the mouse Six genes (Six1/2, Six4/5) revealed
similar target sequence specificities for these mammalian counterparts of
so. Six2, Six4/AREC3 and Six5 effectively bind to the same
target sequence in a DNA fragment called ARE (Atpla1 regulatory element) that
can be found in the Na,K-ATPase alpha1 subunit gene.
Six1 and Six4 have been shown to bind to MEF3 sites in the
myogenin and in the aldolase A muscle-specific (pM) promoters.
Recently, mammalian Six4 has been shown to bind additionally to the
transcriptional regulatory element X (TreX) within the muscle creatine kinase
(MCK) enhancer (Pauli, 2005).
Comparison of all these sites confirms that the three nucleotides
suggested to be the most important for So-DNA interaction are present and
conserved within these motifs (nt. 1, 4 and 9 in the identifed binding site). In the case of the
MEF3 site (which comprises seven nucleotides that include only two of the
nucleotides important for So-DNA interaction), the
original publications were examined to check if the third conserved nucleotide is also
present, and in most of the cases its conservation has been verified. In fact,
there is only one exception published in a study that describes two
Six2 target sites (Pauli, 2005).
Nevertheless, by combining the vast majority of previous studies describing
protein-DNA interaction of Six genes and this study of So-DNA
interaction, it is inferred that So, Six1, Six2, Six4 and Six5
have very similar DNA-binding properties. In the case of so, it is
proposed that the consensus sequence GTAANYNGANAY(C/G) marks a good starting
point for the identification of additional targets of So, thereby helping to
unravel the complex genetic interactions that orchestrate the development of
the visual systems of Drosophila (Pauli, 2005).
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).
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).
Bolwig's organ formation
and atonal expression are controlled by the concerted
function of hedgehog, eyes absent and sine oculis. Bolwig's
organ primordium is first detected as a cluster of about
14 Atonal-positive cells at the posterior edge of the ocular
segment in embryos and hence, atonal expression may
define the region from which a few Atonal-positive founder
cells (future primary photoreceptor cells) are generated by
lateral specification. In Bolwig's organ development, neural
differentiation precedes photoreceptor specification, since
Elav, a neuron-specific antigen, whose expression is under
the control of atonal, is expressed in virtually all early-Atonal-positive cells prior to the establishment of founder
cells. Neither Atonal expression nor Bolwig's organ
formation occurs in the absence of hedgehog, eyes absent
or sine oculis activity. Genetic and histochemical analyses
indicates that (1) the required Hedgehog signals derive from
the ocular segment, (2) Eyes absent and Sine oculis act
downstream of or in parallel with Hedgehog signaling and
(3) the Hedgehog signaling pathway required for Bolwig's
organ development is a new type and lacks Fused kinase
and Cubitus interruptus as downstream components (Suzuki, 2000).
Prior to the establishment of Bolwig's organ founder cells, virtually all Bolwig's organ precursor (BOP) cells acquire neural fate.
The earliest event of Bolwig's organ development may be ato
expression at mid stage 10: this early ato expression defines the
area of BOP. Early ato expression is regulated by the concerted
action of Eya, So and Hh signals. During late stage 10 and early
stage 11, Elav, a neuron-specific antigen, begins to be expressed
in almost all BOP cells. This elav expression is likely to be
regulated by Ato activity, since (1) BOP elav expression is
reduced extensively in ato mutants and (2) the number
of Elav-positive cells at stage 11 and Kr-positive Bolwig's
organ neurons at stage 16 considerably increases upon ato
misexpression. As with ato expression, eya, so and hh activity is
essential for elav expression in BOP cells.
In contrast to elav expression, ato expression is restricted to
three founder cells at stage 12: this late ato
expression disappears by the end of stage 12. Photoreceptor
specification of putative founder cells may start during stage
11, since at late stage 11, 2-3 cells in a cluster start expressing Kr and/or Glass, which are specific markers for larval photoreceptors. Cells expressing Kr and/or Glass increase during stages 12-13 and all 12 photoreceptors express both Kr and Glass by stage 16. Similarly, a peripheral nervous system-specific signal
recognized by mAb22C10 (see Futsch) appears in a few BOP cells at stage
12 and becomes recognizable in all Bolwig's neurons by stage
16. Late ato expression may also be
essential for normal photoreceptor formation. In ato mutants,
neither Kr-positive nor mAb22C10-positive cells can be seen
in stage-16 future larval eyes (Suzuki, 2000).
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).
The Pax-6 gene encodes a transcription factor with two
DNA-binding domains, a paired domain and a homeodomain, and
is expressed during eye morphogenesis and development of
the nervous system. Pax-6 homologs have been isolated
from a wide variety of organisms ranging from flatworms
to humans. Since loss-of-function mutants in insects and
mammals lead to an eyeless phenotype and Pax-6 orthologs
from distantly related species are capable of inducing
ectopic eyes in Drosophila, it has been proposed that Pax-6 is
a universal master control gene for eye morphogenesis. To
determine the extent of evolutionary conservation of the eye
subordinate target genes of Pax-6, subordinate genes of Pax6 have been sought. Expression of two genes, sine oculis (so) and eyes
absent (eya), is induced by eyeless (ey), the Pax-6 homolog
of Drosophila. Evidence from ectopic
expression studies in transgenic flies, from transcription
activation studies in yeast, and from gel shift assays in vitro
is presented supporting the notion that the EY protein activates transcription of sine oculis by
direct interaction with an eye-specific enhancer in the long
intron of the so gene. Sequences of the putative sites are at maximum 88%
homologous (70% with
the consensus PRD binding site sequence). The functional importance of the
eye-specific enhancer of so has been demonstrated in vivo by means
of the so1 mutant, which deletes a 1.3 kb region including the
enhancer, but leaves the coding sequences intact. In so1
homozygous flies, ey is neither capable of inducing so
transcription nor can it induce ectopic eyes. In contrast to ey,
its paralog toy induces both ectopic so transcription and
ectopic eyes in a so1 mutant background. This indicates that
ey and toy regulate so by different mechanisms (Niimi, 1999).
teashirt was initially identified as a gene required for the specification of the trunk segments in Drosophila
embryogenesis and encodes a transcription factor with zinc finger motifs. Targeted
expression of teashirt in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a
phenotype similar to that produced from targeted expression of eyeless, dachshund, and eyes absent. The expression of so and dac are induced in the antennal disc by the ectopic expression of tsh, suggesting that tsh may act
upstream of these genes in eye development.
Furthermore, teashirt and eyeless induce the expression of one another, suggesting that teashirt is part of the
gene network that functions to specify eye identity (Pan, 1998).
However, these results do not prove that tsh does
play a role in specifying the eye identity during normal development. To address this issue, an examination was carried out to see if tsh is expressed at the right time and the right place to have a role in specifying the eye
identity. Indeed, TSH mRNA is expressed in the eye disc, with the strongest expression anterior to the
morphogenetic furrow. This pattern of expression is similar to that of ey, a gene that is
known to play an essential role in specifying eye identity. An examination was carried out to see if loss-of-function
mutations of tsh affect eye development. Several weak loss-of-function tsh alleles were examined and no eye defects were found. X-ray-induced mitotic recombination was used to generate mutant clones of a null tsh allele.
tsh mutant clones were recovered at a frequency similar to the wild-type control, and sections through
the mutant clones revealed a normal ommatidial organization. These data suggest that tsh may
play a redundant role during normal eye development, and the requirement for tsh may be masked by
other factor(s) that play a role similar to tsh (Pan, 1998).
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 zerknuellt (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:
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).
Drosophila eye development is controlled by a conserved network of
retinal determination (RD) genes. The RD genes encode nuclear proteins that
form complexes and function in concert with extracellular signal-regulated
transcription factors. Identification of the genomic regulatory elements that
govern the eye-specific expression of the RD genes will allow a better
understanding of how spatial and temporal control of gene expression occurs during
early eye development. Conserved non-coding sequences (CNCSs)
between five Drosophilids were compared along the ~40 kb genomic locus of the RD gene dachshund (dac). This analysis uncovers two separate eye
enhancers, in intron eight and the 3' non-coding regions of the
dac locus, defined by clusters of highly conserved sequences. Loss-
and gain-of-function analyses suggest that the 3' eye enhancer is
synergistically activated by a combination of eya, so and
dpp signaling, and only indirectly activated by ey, whereas
the 5' eye enhancer is primarily regulated by ey, acting in
concert with eya and so. Disrupting conserved So-binding
sites in the 3' eye enhancer prevents reporter expression in vivo. These
results suggest that the two eye enhancers act redundantly and in concert with
each other to integrate distinct upstream inputs and direct the eye-specific
expression of dac (Anderson, 2006).
The smallest fragment in the 3' dac eye enhancer that can
respond to dpp, eya and so is 3EE194 bp,
which is centered around two CNCS blocks of ~40 bp and 20 bp. These two CNCS
blocks are also common to all active fragments of the 3' eye enhancer.
These two evolutionarily conserved stretches were scanned for known, genetically
upstream transcription factor binding sites. The 40 bp conserved
stretch contains two putative consensus So-binding sites, S1-5'-CGATAT
and S2-5'-CGATAC, compared with the consensus 5'-(C/T)GATA(C/T)
described previously. Each of these putative So-binding sites in 3EE were mutated individually and in combination to test their requirement for normal enhancer activity in vivo. Mutation of individual So-binding sites causes a severe reduction, but not complete elimination, of enhancer activity in vivo. However,
simultaneous mutation of both So binding sites completely abolishes enhancer
activity in vivo. These results, coupled with loss-and gain-of-function analyses with dpp, eya and so, suggest that So binds to the 3' eye enhancer
directly and nucleates a protein complex that includes Eya to regulate
3EE. However, despite much effort using a wide variety of binding
conditions, it was not possible to demonstrate specific, direct binding of So
protein to oligos that contain these So-binding sites. The
5' eye enhancer, which has four CNCS blocks, were scanned for potential upstream
transcription factor binding sites and no strong candidate binding sites
were found within the CNCS blocks (Anderson, 2006).
Loss- and gain-of-function analyses with the two eye enhancers suggest that
each enhancer is regulated by a distinct set of protein complexes. The
5' eye enhancer is activated by a combination of ey, eya and
so, but is not activated by Dpp signaling. 5EE is activated
by ectopic ey expression even in eya and so
mutants, suggesting that it is regulated exclusively by ey. However,
somewhat paradoxically, expression of 5EE, the intron 8 enhancer, is lost in eya and so mutants even though ectopic expression of a combination of
dpp, eya and so does not activate this enhancer.
Furthermore, driving high levels of ey in so1
mutant eye discs restores 5EE-lacZ expression. Coupled together,
these results suggest that 5EE is primarily regulated by ey
but that the regulation of 5EE by ey also requires
eya and so (Anderson, 2006).
By contrast, the 3' dac eye enhancer is regulated by a
combination of eya, so and dpp signaling, but is not
directly dependent on ey. 3EE-GFP expression is lost in
eya2 and so1 mutant eye discs, and in
posterior margin mad1-2 mutant clones. Furthermore,
ey cannot bypass the requirement for eya and so to
activate 3EE. Conversely, 3EE is strongly induced by
co-expression of eya and so. Moreover, dpp
signaling via the tkv receptor can synergize with eya and
so to induce 3EE in ectopic expression assays. Furthermore,
neither Mad nor Medea, the intracellular transducers of Dpp
signaling, is sufficient to bypass the requirement for activation of the Dpp
receptor Tkv in these assays. Thus, it is concluded that events
downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are
essential for the synergistic activation of the 3' dac eye
enhancer by eya and so. Taken together, these results
suggest that there are distinct requirements for the activation of the
5' and 3' dac eye enhancers. However, the exact nature of
the protein complexes that regulate 5EE and 3EE remain to be
determined (Anderson, 2006).
Morphogenetic furrow (MF) initiation is completely blocked in posterior margin
dac3-null mutant clones. However, dac3
clones that do not include any part of the posterior margin develop and do not
prevent MF progression, but do cause defects in ommatidial cell number and
organization. This dichotomy in dac function is reflected in the
two eye enhancers characterized in this study. Analysis of
dac7 homozygotes demonstrates that the 3' eye
enhancer is dispensable for MF initiation and progression. It is proposed that in
dac7 mutants, the intact 5EE enhancer is
sufficiently activated by ey to drive high enough levels of
dac expression to initiate and complete retinal morphogenesis.
However, dac7 mutants have readily observable defects in
ommatidial organization. Thus, it is further proposed that this lack of normal
patterning in dac7 mutants is most likely due to the loss
of 3EE, which normally acts in concert with 5EE after MF
initiation, to integrate patterning inputs from extracellular signaling
molecules such as Dpp with tissue-specific upstream regulators such as ey,
eya and so. However, it is not known if the 3' eye enhancer
is sufficient to initiate dac expression in the absence of the
5' eye enhancer (Anderson, 2006).
Based on the results, a two-step model is proposed for the regulation of
dac expression in the eye. First, the initiation of dac
expression in the eye disc is dependent on Ey binding to 5EE.
However, Ey is fully functional only when So and Eya are present. It is
possible that Ey recruits So and Eya to 5EE, but a model is favored in
which Ey bound to 5EE cooperates with an So/Eya complex bound to
3EE to initiate dac expression in the eye. After initiation
of the MF, dac expression is maintained by an Eya and So complex
bound to 3EE. In addition, 3EE can integrate patterning
information received via dpp signaling, thereby allowing the precise
spatial and temporal expression of dac in the eye. This two part
retinal enhancer ensures that dac expression is initiated only after
ey activates eya and so expression. Thus, the
dac eye enhancers provide a unique model with which the sequential
activation of RD proteins allows the progressive formation of specialized
protein complexes that can activate retinal specific genes (Anderson, 2006).
The redundancy in dac enhancer activity also explains the
inability to isolate eye-specific alleles of dac, despite multiple
genetic screens. The modular nature of
the two enhancers and their potential ability to act independently or in
concert suggest that both enhancers must be disrupted to block high levels of
transcription of dac. Thus, two independent hits in the same
generation, a phenomenon that occurs infrequently in genetic screens, would be
required to obtain an eye-specific allele in dac (Anderson, 2006).
Despite much investigation, very few direct targets of RD proteins,
especially for Eya and So, have been identified. One study suggests that So
can bind to and regulate an eye-specific enhancer of the lz gene. However,
lz is not expressed early during eye development and is required only
for differentiation of individual cell types. The
results suggest that regulation of dac expression occurs via the
interaction of two independent eye enhancers that are likely to be bound by
Ey, Eya and So, and respond to dpp signaling. This analysis of the
3' eye enhancer suggests that two putative conserved So-binding sites
are essential for 3EE activity in vivo. Mutation of individual
So-binding sites dramatically reduces, but does not completely eliminate,
reporter expression in the eye. Mutating both predicted So-binding sites
completely blocks enhancer activity in vivo. Thus, it is concluded that So binds
to 3EE via these conserved binding sites. However, it has not been
possible to demonstrate a direct specific interaction of either So alone or a
combination of Eya and So with oligos that contain these putative So-binding
sites in vitro. It is possible that other unidentified proteins are required
for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that
responds to ectopic expression of dpp, eya, and so contains
no conserved or predicted Mad-binding sites. This raises the intriguing
possibility that dpp signaling activates other genes, which then
directly act with eya and so to regulate the 3' eye
enhancer. Alternatively, a large complex that includes Eya, So and the
intracellular transducers of dpp signaling, such as Mad and Medea,
may be responsible for activation of 3EE. Similarly, the results
suggest that the 5' eye enhancer is regulated primarily by ey.
However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey
is fully functional only in the presence of Eya and So. Thus, Ey either
independently recruits Eya and So into a 5' complex or is activated by
virtue of its proximity to the So/Eya complex bound to the 3' enhancer
or both (Anderson, 2006).
The exact order and dynamics of protein complex assembly at 5EE
and 3EE requires further investigation. However, the two dac
eye enhancers are extremely useful tools with which to investigate fundamental
issues about the mechanism of RD protein action. One significant issue
concerns the mechanism of Eya function during eye development. Eya consists of
two major conserved domains, an N-terminal domain that has phosphatase
activity in vitro and a C-terminal domain that can function as a
transactivator in cell culture assays. So
contains a conserved Six domain and a DNA binding homeodomain.
However, it is unclear if Eya provides phosphatase activity, transactivator
function, or both, in this complex. Characterization of the components of the
protein complexes that regulates dac expression may uncover the
targets of Eya phosphatase activity during eye development. Thus, the
isolation of two eye enhancers with distinct regulation provides very useful
tools with which to study protein complex formation and function during
Drosophila retinal specification and determination (Anderson, 2006).
During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of 'eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. This study shows that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. These transcription factors interact in vitro and indirect evidence is provided that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation (Zhang, 2006).
This study found that regulatory elements controlling the early phase of ato expression in the eye lie within a 1.2 kb region located 3.1 kb downstream of the ato transcription unit. The early phase of ato
transcription results from the integration of multiple regulatory inputs
through separate cis-regulatory modules present within the 1.2 kb region (Zhang, 2006).
Cis-regulatory elements essential for gene activation map to the last 348
bp of the 1.2 kb region and include the So- and Ey-binding sites.
Interestingly, the 348 bp region contains two relatively large (A1=99 bp and A2=140 bp) DNA sequences that are highly conserved from D. melanogaster to D. virilis. Based on this observation, constructs were generated containing only A1 or A2. However, neither region alone was sufficient to drive the stripe of reporter gene expression in the eye disc. Based on these results, it is conclude that the 348 bp region constitutes a 'core' or 'minimal' enhancer region for the transcriptional activation of ato in eye progenitor cells.
Other factors undoubtedly bind to sequences within A1-A2 and regulate gene
expression as neither A1 nor A2 alone are sufficient to drive expression in
the eye disc. Genetic evidence suggests that signaling by the Bmp4-type factor
Decapentaplegic (Dpp) also contributes to ato activation and two
putative binding sites for Mad (a transcription factor shown to activate Dpp
pathway targets) appear to be required for ato expression in all
discs. However, a Mad consensus site present in the A2 box does not correspond to either of the two elements previously identified . Moreover,
both the previously identified sites lie within the L fragment well upstream
of the M'-M" interval containing the eye-disc enhancers. Future analyses of 3' enhancer-promoter interactions may resolve this issue (Zhang, 2006).
Separate cis-regulatory elements located within the conserved DNA regions
IC1 and IC2 (IC1=88 bp and IC2=133 bp) control initial clusters formation. This feature of ato expression has been shown to require Notch (N) function. Sequence analysis of
the IC1-IC2 region does identify a binding site for the effector of N
signaling Suppressor of Hairless [Su(H)]. However, contradictory reports have
been published on how Notch controls ato expression. Sun and
colleagues found that transcription of ato is uniformly upregulated upon inactivation of Notch in Nts1 mutant discs. By contrast, early Ato protein expression is severely reduced in null Notch mutant clones . Since
these experiments made use of different genetic reagents, it is difficult to
interpret these results. Notch signaling may independently regulate
ato expression at the mRNA and protein levels. Alternatively, the
source of the discrepancy may lie in the use of different alleles, one
hypomorphic (Nts1), and the other null
(N54l9) (Zhang, 2006).
Lastly, activation of the
3'ato348-ßgal reporter (core
element) occurs prematurely as compared with endogenous ato. The
3'ato348-ßgal mRNA is also
found in cells lying just anterior to the proneural domain. Eye progenitors
from this region are at a developmental stage referred to as pre-proneural and
are characterized by the expression of the transcription factor Hairy (H) in
addition to RD proteins. In the absence of Hairy and its partner Extra
Macrochaetae, neurogenesis begins precociously within the eye disc. Thus,
Hairy contributes to the downregulation of ato expression and
prevents precocious neurogenesis. Activation of the reporters
3'ato348-ßgal and
3'ato488-ßgal (but not
3'ato1.2-ßgal or
3'ato1.2-Δ298-ßgal) in
pre-proneural cells suggests that cis-elements mediating anterior repression
lie within the 1.2 kb DNA fragment but outside the IC and A boxes. Although a
search for canonical Hairy-binding sites does not identify potential
regulatory elements, additional short stretches of evolutionarily conserved
DNA are present and may contribute to this and/or other aspects of
ato regulation (Zhang, 2006).
Over the last few years, Ey and So have been shown to play a crucial role
in the deployment and maintenance of the RD network by directly regulating the
transcription of several eye-specification genes [ey, so, eya,
dachshund (dac) and optix]. However, little is known about downstream targets of the RD cascade. Although So also activates the post-MF expression of hedgehog and lozenge, this gene regulation is likely to reflect the late, differentiation-related functions of So. Thus, it is unclear how the RD factors induce eye formation and what aspects of the morphogenetic program they control directly (Zhang, 2006).
The results strongly suggest that the transcription factors Ey and So
control activation of ato expression. This is the first example of a
gene required during eye morphogenesis that is directly regulated by the RD
network. The direct control of ato by Ey and So is a likely reason
why ectopic eye induction by Eya+So or Dac depends on the activation of their
upstream regulator ey. Other downstream targets may also be similarly controlled by multiple RD factors (Zhang, 2006).
The in vitro and in vivo evidence presented in this study also suggest that Ey and So may form a complex when bound to the adjacent cis-regulatory sites in the 3'ato core element. Together with the
previously reported interactions of Eya-So and Eya-Dac, this
finding raises the possibility that additional multimeric complexes involving
several RD factors may also be involved in driving the transcriptional program
for eye development. The observation that normal eye development is severely disrupted when one or
another RD factor is over-expressed suggests that the RD proteins must be
present at an appropriate level relative to one another. As all four proteins,
Ey, Eya, So and Dac, have now been shown to interact in various combinations,
the formation of such complexes and the recruitment of additional shared
co-factors are likely to be sensitive to the relative concentration of RD
factors present in eye progenitor cells (Zhang, 2006).
The model of gene regulation exemplified by the control of ato
transcription provides a strong rationale for the feedback regulatory loops
that link late and early RD gene expression. This regulation is likely to play
a crucial role in ensuring the presence of appropriate levels of all four RD
factors to optimize complex formation and co-regulation of downstream
targets (Zhang, 2006).
Current models for the co-ordination of
organ identity and neurogenesis in the eye place the Pax6 pathway either
upstream of, or in parallel to, the control of neurogenesis. The findings
presented in this paper favor the former model. Separate
regions have been identified for the regulation of ato transcription in the eye versus
other sensory organs (JO and CH). In addition, the presence of Ey- and
So-binding sites that are required in vivo for reporter gene activation
strongly suggests that endogenous ato expression is directly
regulated by these factors. Thus, the RD network does not merely modify
sensory organ development within the eye disc, but does, in fact, directly
control it. In doing
so, it also contributes to the co-ordination of selector and neurogenic inputs
required to generate complex sensory structures such as the eye (Zhang, 2006).
Is this regulatory relationship between Ey-So and ato ancestrally
derived? That is, was the direct link between ancestral Pax- and Ath-like
genes already established in the protosensory organ that gave rise to today's
ato-dependent sensory structures? The association of Pax-, Six- and
Ath-type factors with sensory perception is not restricted to photic sensation
but extends to mechanoreception in diverse organisms including mouse,
jellyfish and mollusks.
In the jellyfish P. carnea, which lacks eyes but responds to a
variety of environmental stimuli including light, expression of a putatively
ancestral-like PaxB gene, Six1/2, Six3/6 and atonal-like
1 is associated with neuronal precursors found in the medusa tentacles.
Although the studies carried out in more basal metazoa consist mostly of
analyses of gene expression and not function, this evidence does suggest that
the association of Pax/Six/Ath-type factors and sensory organ development is
ancient and may have been retained over more than 600 million years of
evolutionary history (Zhang, 2006).
It is possible that the mechanisms of transcriptional regulation uncovered
between Pax and Six genes and
between Pax/Six and ato may have arisen early
during evolution. Such regulatory interactions may have favored the continued
association of Pax/Six/Ath as various modifications of their genetic
cascades led to the development of more complex and diverse sensory organs.
The investigation of ato/Ath gene regulation in other sensory organs
and in basal metazoans is likely to clarify the evolutionary relationship
among these pathways and the sensory modalities they control (Zhang, 2006).
So and Eyes absent are able to physically interact through their evolutionarily conserved domains. The sequences responsible for interaction are localized to the N-terminal domain of Eya, while the So interaction domain is localized to the Six domain, a conserved sequence shared with vertebrate So homologs. Because of the multiple effects of the So/Eya interaction in MF induction, cell proliferation and neural induction, it is proposed that a So/Eya complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity (Pignoni. 1997).
The eyes absent gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by
programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and
physically interacts in vitro with two other proteins of eye development, Sine oculis and Dachshund. The Eya protein sequence,
while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, eya alleles have been sequenced. Loss of the entire Eya Domain is null for eya activity, but alleles with
truncations within the Eya Domain display partial function. The molecular genetic analysis was extended to interactions within the Eya Domain. This analysis has
revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminish the interactions
with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions
within it (Bui, 2000).
This analysis of the mutations in the Eya Domain was extended to the situation in vivo by generating transgenics expressing the selective point mutants that disrupt interactions with So and Dac in the yeast two-hybrid system. Although this has failed to provide evidence in support of a special functional relevance of the Dac interaction (both mutant forms appeared to interact similarly in ectopic eye formation upon coexpression with Dac) evidence has been found supporting the importance of the So interaction. These data indicate that the EyaE11 mutant form shows a diminished ability to synergize with So upon coexpression. This supports the hypothesis that the eyaE11 mutation within the Eya Domain disrupts interactions in vivo with so (and/or possibly with other Six homologs) that are critical for the function in eye formation. The EyaE7 mutant form, which shows a disrupted Dac interaction, still supports ectopic eye formation, although at decreased penetrance compared to normal Eya. dac null mutations frequently show some degree of eye development, suggesting that dac may be partially redundant in eye formation. Therefore, even if interaction with Dac in vivo were disrupted by the eyaE7 mutation, eye formation might still occur due to compensation by such mechanisms. Nevertheless, this eya allele also shows a dominant reduced-eye phenotype when coexpressed with so -- this is a new property not observed with the wild-type Eya protein. The eyaE7 mutation may generate a protein with some dominant-negative property in eye formation. The data that So and Dac may interact, in part, differentially within the conserved domain of Eya supports the idea that the three proteins have the potential to interact in a single complex in vivo. Such a hypothesis, however, is complicated by other data indicating that the molecular activity of Eya-So coexpression in eye formation is at least in part distinct from that of Dac or Eya-Dac coexpression: whereas Dac, and Eya coexpression with Dac, activate an eya enhancer, Eya alone or Eya with So fails to activate enhancer activity, despite ectopic eye formation (Bui, 2000).
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