eyeless
An intron of the eyeless (ey) gene contains an enhancer that regulates the eye specific expression of the gene in the eye disc primordia of embryos and in the eye imaginal discs of third instar larvae. The enhancer is located in the intron preceding exon 3 of ey. Moreover, a 212-bp enhancer element is necessary and sufficient for the enhancer function. It is partially conserved in Drosophila hydei and contains putative Pax-6 Paired domain binding sites. Several binding sites are required for the eye specific expression, and, therefore, it is proposed that a Pax-6-like molecule is a positive transactivator for the eye specific ey expression. This transactivator recently has been identified as twin of eyeless, the second Pax-6 gene in Drosophila (Hauck, 1999).
The Drosophila Pax-6 homologs eyeless and twin of eyeless are expressed in the eyes and in the central nervous system (CNS). In addition to the pivotal functions in eye development, previous studies have revealed that ey also plays important roles in axonal development of the mushroom bodies, centers for associative learning and memory. It has been reported that a second intron enhancer that contains several Pax-6 binding sites mainly controls the eye-specific expression, but the DNA sequences that control CNS expression are unknown. In this work, the transcriptional enhancer elements of the ey gene that are required for the CNS expression in various developmental stages have been dissected. CNS expression is independent of the eye-specific enhancer of the second intron. By systematic reporter studies, several discrete DNA elements in the 5' upstream region and in the second intron have been identified that cooperatively interact to generate most of the ey expression pattern in the CNS. DNA sequence comparison between the ey genes of distant Drosophila species has identified conserved modules that might be bound by the upstream regulatory factors of the ey gene in CNS development. Furthermore, by RNA interference and mutant studies, it has been shown that ey expression in the brain is independent of the activity of toy and ey itself whereas in the eye primordia it requires both, supporting the notion that ey and toy are regulated by parallel and independent regulatory cascades in brain development (Adachi, 2003).
The expression of Ey in the CNS is first detected in the ventral nerve cord (VNC) at stage 9 and in the early brain at stage 10. After germ band retraction, several clusters of Ey-expressing cells are observed in the embryonic brain. Ey is expressed in several clusters of cells in each of the embryonic brain neuromeres (b1, b2 and b3). In the first neuromere b1, a prominent cluster of Ey-expressing cells is observed at the most anterior region (in neuraxis), which gives rise to the MBs. Additional groups of cells are located dorsally (b1D), ventrally (b1V) and medio-laterally (brackets). In contrast, Ey is not expressed in the optic lobe primordia. In the ventral neuromeres Ey is expressed in a large segmental pair of cells located laterally as well as in a few pairs of cells located more medially. Later in the third instar larval stage Ey is expressed in MBs, in the medial part of the optic lobes and in segmental pairs of cells in the VNC. In the larval MBs, Ey is strongly expressed in all MB neurons. After metamorphosis, Ey expression is detected in many clusters of neural cells in the adult brain. Strong Ey expression continues in the Kenyon cells located in the posterior-dorsal region of the adult brain (Adachi, 2003).
A GAL4 enhancer trap, OK107, was initially isolated as an enhancer trap line that shows strong MB expression in the adult brain. Its P-element is inserted on chromosome 4, where ey is located. By PCR and sequencing, the exact location of the OK107 insertion was found to be 6.5 kb upstream of the first exon of ey. OK107 is confirmed to be expressed in the embryonic MB primordia as well as the larval MBs. Double immunostaining verified colocalization of OK107 expression and Ey in the brain, in that all GAL4-expressing neurons in OK107 indeed express Ey, although many Ey-expressing cells in other regions of the CNS do not express OK107 GAL4. In MBs, OK107 faithfully recapitulates the Ey expression throughout brain development. The coexpression of OK107 and EY in MBs was further confirmed in a complementary experiment, in which OK107 was used to drive a dominant-negative form of Ey. The phenotypes observed in this experiment reproduce the range of MB phenotypes observed in ey mutants (Adachi, 2003 and references therein).
Previous work led to the identification of the eye-specific enhancer at the 3' end of the second intron. Despite its robust transcriptional activity in the eye,
minimal eye fragments such as 5D12 and D02 lack the potential to drive the LacZ reporter in the CNS, highlighting distinct requirements of cis-regulatory regions in the eye and CNS. In contrast, the E3.6 intronic fragment is able to drive CNS expression in reversed orientation, indicating a neural enhancer at the 5' end of the second intron, where conserved CNS motifs are confirmed (Adachi, 2003).
Apart from the intronic enhancer, a 5 kb upstream sequence drives strong LacZ expression in the CNS, including the MBs. This result is consistent with the robust MB activity of the GAL4 enhancer trap OK107, which is inserted 6.5 kb upstream of the first exon. Intriguingly, deletion analysis of the upstream region results in complex expression patterns that only partially overlap with the endogenous Ey pattern, with occasional ectopic activation of LacZ expression. Thus, these results argue for the existence of both positive and negative enhancer modules in the upstream and intronic sequences that cooperatively define most of the developmental expression pattern of ey in the CNS (Adachi, 2003).
However, the LacZ expression of ey12ER does not faithfully represent the endogenous ey patterns. This is particularly evident in the embryonic brain, in which expression of ey12ER is detected in virtually all the Ey positive neurons but its strength is not proportional to the endogenous Ey levels. Perdurance of LacZ protein might account for part of this discrepancy. Alternatively, it could be that additional modular elements are required to further refine the expression levels in different neurons. It is also noteworthy that none of the constructs drives LacZ expression in the pars intercerebralis, lateral horns and lateral neurons in the adult brain. These results suggest that enhancers for the expression of these adult neurons might be missing in the current series of reporter constructs. Indeed, it has been shown that regulatory elements located at the 3' end of the mouse Pax-6 gene control a part of expression in the developing pretectum, neural retina and olfactory region. Another set of reporter constructs and/or novel 3' enhancer trap lines are required to clarify this point (Adachi, 2003).
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).
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).
Mutations in four other Drosophila genes (eyes absent, sine oculis, eyes gone and eyelisch) have similar phenotypes but do not affect the expression pattern of eyeless, indicating that these genes are downstream or act in parallel with eyeless (Cheyelle, 1994).
Ectopic expression of sine oculis has little or no effect on antennal, wing, or leg disc development, while ectopic eyes absent expression often causes mild growth alterations resulting in extra folds in the epithelium and, rarely, formation of small ectopic ommatidial arrays in the antennal disc. Coexpression of so and eya leads to a dramatic increase in the development of ectopic eye tissue in antennal discs. These ommatidial arrays lead to adult eye structures. Ectopic so/eya induce eyeless expression in the antennal disc. When expressed in eyeless mutant discs, ectopic so/eya produces growth alterations, but ectopic eyes are not observed (Pignoni, 1997).
The Notch signaling pathway defines an evolutionarily conserved cell-cell interaction mechanism that throughout development controls the ability of precursor cells to respond to developmental signals. Notch signaling regulates the expression of the master control genes eyeless, vestigial, and Distal-less, which in combination with homeotic genes induce the formation of eyes, wings, antennae, and legs. Therefore, Notch is involved in a common regulatory pathway for the determination of the various Drosophila appendages (Kurata, 2000).
The intracellular domain of the truncated Notch receptor reflects a constitutively activated state (Notch activated, Nact) and the extracellular domain of the truncated receptor mimics loss-of-function phenotypes representing a dominant negative form (Notch dominant negative, Ndn). To examine the role of Notch signaling in early eye development, these truncated forms were expressed in the early eye imaginal disc, using the GAL4 system with the eye-specific enhancer of the ey gene. This eye-specific enhancer induces target gene expression in the eye primordia of the embryo and maintains expression throughout eye morphogenesis. In contrast to ey expression in the wild-type eye-antennal disc, the enhancer-driven reporter gene expression is not down-regulated in the differentiating cells posterior to the morphogenetic furrow but it extends all over the eye disc and into the area of the antennal disc where the rostral membrane is going to be formed. However, the expression in the antennal disc is quite variable from disc to disc. Consistent with previous loss of Notch function studies, crossing ey enhancer-GAL4 (ey-GAL4) flies to a stock carrying Ndn under an upstream-activating sequence for GAL4 (UAS-Ndn) results in a strongly reduced eye phenotype in all transheterozygous flies similar to that of the ey2 mutant. Inhibition of Notch signaling by misexpression of Hairless (H) and dominant negative forms of Delta (Dl) and Serrate (Ser) also leads to a reduction or complete absence of the eye (Kurata, 2000).
Activation of Notch signaling by crossing ey-GAL4 flies to a UAS-Nact line leads to significant pupal lethality, but all transheterozygotes that escape lethality show hyperplasia of the eyes with a significant increase in the number of facets. The disc overgrowth is found in all eye discs of ey-GAL4 UAS-Nact larvae, consistent with a role for Notch signaling in growth control of the eye imaginal disc. Furthermore, about 16% of the escapers form ectopic eyes on the rostral membrane of the head, which is derived from the antennal disc (Kurata, 2000).
Immunostaining of eye-antennal discs of ey-GAL4 UAS-lacZ UAS-Nact larvae using an ELAV antibody to identify the differentiating photoreceptor cells and a beta-galactosidase antibody to monitor the Nact protein shows that both the strong hyperplasia of the eye disc and ectopic eye formation in the antennal disc correlate with the expression of Nact. However, the time window for expression of the truncated receptors is critical. Transheterozygotes in which either Ndn or Nact were driven by the glass promoter GMR-GAL4, which drives expression in all cells posterior to the furrow only, show only a mild phenotypic effect. Ndn results in a roughening of the eye, whereas Nact produces a polished eye phenotype. Therefore, the timing of Notch signaling is of crucial importance (Kurata, 2000).
The reduced eye phenotype caused by expression of Ndn and the induction of ectopic eyes by the expression of Nact are similar not only to loss-and-gain mutants of ey but also resemble two other mutations acting downstream in the ey developmental pathway, eyes absent (eya) and dachshund (dac). Furthermore, a second Pax-6 gene of Drosophila, twin of eyeless (toy), was found to be an upstream regulator of ey capable of inducing ectopic eyes by inducing ey. To determine the epistatic relationship of Notch to those genes, the effect of Ndn on ectopic eye induction by ey and toy was studied (Kurata, 2000).
A dpp-enhancer GAL4 line (dpp-GAL4) was crossed to flies carrying both UAS-Ndn and UAS-ey or alternatively to UAS-Ndn and UAS-toy. Transheterozygotes from both crosses exhibit ectopic eyes on legs and wings in all flies. The size of the ectopic eyes is similar to those of the transheterozygous controls dpp-GAL4 UAS-ey and dpp-GAL4 UAS-toy, respectively, suggesting that Notch acts upstream of ey and toy. Double immunostaining of eye-antennal discs from transheterozygous ey-GAL4 UAS-Nact, UAS-lacZ using an anti-EY antibody to reveal EY protein and anti-beta-galactosidase antibody to indirectly reveal Nact demonstrates that ey expression is induced in all eye discs by the activation of Notch signaling. Moreover, strong ectopic expression of Ey protein has been observed. The ectopic expression pattern of Ey corresponds to that of lacZ reflecting the expression of Nact protein. Analysis of ey expression by in situ hybridization indicates that ey is induced at the transcriptional level. Similarly, ectopic expression of toy also is induced in the antennal discs of ey-GAL4 UAS-Nact larvae. Thus, activation of Notch signaling can induce toy and ey expression in antennal discs. Expression of Nact also correlates with the ectopic induction of photoreceptor cells as revealed by ELAV staining (Kurata, 2000).
Notch activation of ey and toy depends on the downstream effector of Notch, Suppressor of Hairless, because Su(H) mutant clones generated anterior to the morphogenetic furrow in the eye fail to produce adult structures, in agreement with a requirement for Notch signaling during eye morphogenesis (Kurata, 2000).
Consistent with the finding that ey acts downstream of Notch, the expression of Nact in an ey2 or eyR hypomorphic mutant background generates eyes of a reduced size. Approximately 72% of the ey-GAL4 UAS-Nact; ey2 flies that survived were found to have reduced eyes and about 15% of these flies had both a reduced original and a reduced ectopic eye. However, in addition to ectopic eyes Nact also induces ectopic antennae in 25% of these flies on the side of the head that is derived from the eye disc. Many of the induced ectopic antennae were complete with all three antennal segments and the arista (Kurata, 2000).
Based on these findings, a model is proposed to explain the difference between the eye and antennal pathway starting from a common signaling mechanism. Notch signaling induces the expression of both ey and Dll. However, in the eye primordia ey represses Dll and induces eye morphogenesis. By contrast, in the antennal disc ey is repressed by a repressor, resulting in Dll expression that confers antennal (ventral appendage) specificity. Two of the possible candidates for the repressor are the homeobox genes exd and hth, because both exd- and hth- mutant clones in the rostral membrane region of the antennal disc can result in ectopic eye development, which presumably is caused by derepression of ey. Both exd and hth also may function in conjunction with Dll, serving as corepressors. The present study extends the fundamental role of Notch by indicating that the implementation of entire developmental programs leading to appendage formation and organogenesis may be controlled by Notch activity (Kurata, 2000).
The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. Mutations in axin prevent photoreceptor differentiation and leads to tissue overgrowth; both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. Signaling by Dpp and Hh normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Dpp signaling can promote Ey downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors (Lee, 2001).
Two characteristics distinguish anterior from posterior behavior in the eye disc: growth occurs in the anterior, with the exception of the second mitotic wave, and differentiation occurs in the posterior. Wg signaling regulates both of these properties. Wg signaling promotes the growth of eye disc cells. Loss of axin causes dramatic overgrowth and outgrowth of cells in the eye disc, and this phenotype is due only to excessive Wg pathway activity, since it can be blocked by a dominant negative form of dTCF/Pangolin. The strength of the phenotype may reflect higher levels of Wg signaling than are induced by loss of sgg; perhaps Axin contributes to retaining Arm in the cytoplasm, in addition to promoting its phosphorylation. Vertebrate Axin has been shown to associate with mitogen-activated protein kinase kinase kinase 1 and activate the c-jun N-terminal kinase (JNK) pathway. However, JNK signaling does not appear to be essential for the growth or differentiation of cells in the Drosophila eye disc, and it does not contribute to the axin mutant phenotype in the eye. The ability of Wg signaling to promote overgrowth in the eye disc is consistent with the reduction in the size of the eye disc caused by loss of Wg signaling (Lee, 2001).
Although Wg signaling is sufficient to establish but not necessary to maintain anterior identity in the eye disc, Dpp signaling has complementary properties; it is sufficient to promote but not essential to maintain posterior identity. Ectopic Dpp can downregulate ey in the anterior, but loss of components of the Dpp pathway has a variable effect on ey expression and does not lead to hairless expression or induce reorientation of the furrow in neighboring cells. It is possible that this is due to redundancy with Hh signaling, which also has a weak and variable effect on ey expression. Clones that are mutant for cell-autonomous components of both the Hh and Dpp pathways do not differentiate, while loss of one or the other pathway only delays differentiation; however, even loss of both pathways does not reorient adjacent wild-type cells (Lee, 2001).
Downregulation of ey and photoreceptor differentiation appear to be independent events, since smo clones have a weaker effect on ey expression than Mad clones, despite their stronger effect on differentiation. Anterior Dpp can also downregulate ey over a much longer range than that over which it promotes photoreceptor differentiation, and loss of slimb downregulates ey without leading to ectopic differentiation (Lee, 2001).
The complementary effects of Wg and Dpp on AP polarity appear to be independent of one another. The effect of Dpp is not simply due to its repression of wg, and posterior Wg signaling can upregulate ey even when the Dpp pathway is also activated. However, activation of both pathways reveals the existence of another mechanism that distinguishes anterior from posterior, since the anterior of the disc is more sensitive to the effects of Dpp and the posterior is more sensitive to the effects of Wg. This leads to a striking repolarization of the eye disc when both pathways are activated, resulting in initiation of an ectopic morphogenetic furrow from the anterior margin as well as reduction or elimination of the normal posterior furrow. Since normal development requires anterior cells to be gradually converted to posterior by the action of Dpp and Hh, it is important for Dpp to overcome the effects of Wg in this region. This also underscores the importance of establishing the early expression patterns of wg and dpp (Lee, 2001).
Wg has three roles in early eye disc development: establishment of anterior identity, establishment of dorsal identity, and promotion of growth. Prior to furrow initiation, Pnr, expressed at the dorsal margin, activates wg expression in a broader domain; Wg then activates mirr and the other Iro-C genes throughout the dorsal compartment. Hh may contribute to the activation of these genes through Wg or act independently of Pnr. Upd, which is present at the optic stalk also contributes to the ventral repression of mirr. Mirr represses fng, forming a boundary of fng expression at the DV midline that leads to activation of N in this region. During furrow progression, Hh is expressed in the differentiating photoreceptors and Dpp in a stripe in the morphogenetic furrow. These two signals act to downregulate genes expressed in the anterior such as ey, and allow an anterior to posterior transition. Wg establishes the anterior state, probably at an earlier stage, while another factor (X) contributes to its maintenance. Other factors are necessary to modify the response to Wg in order to determine which cell fate should be specified; this is consistent with data suggesting that Wg signaling alters chromatin structure to allow access to transcription factors. A requirement for multiple signaling systems also ensures accuracy in cell fate determination (Lee, 2001).
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. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). The similarities between vertebrate systems and Drosophila are discussed with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling (Chang, 2001).
Loss of hh results in a strong reduction of the head midline epidermis, a reduction in the size of the brain and optic lobe, and the total absence of the larval and adult eye primordium. Temperature-sensitive shift experiments of hhts2 embryos indicate that the phenocritical period for Hh function in Bolwig's organ development is between 4 and 7 hours. Aside from the larval eye, the primordium of the compound eye, which is marked from stage 12 onward by the expression of eyeless (ey), is also affected by the loss of hh. Heatshock induced overexpression of hh, as well as loss of ptc, causes an increase in larval eye neurons and optic lobe precursors. Interestingly, ectopic Hh activity is able to induce optic lobe and Bolwig's organ tissue in the head midline and thereby generate a cyclops phenotype similar to the condition described above for partial reduction of dpp. Applying heatshocks at different times of development indicates that the phenocritical period for the Hh induced cyclops is early, between 2.5 and 5 hours. Thus, heat pulses administered during this time cause fusion of the optic lobe and, at a lower frequency, of the larval eye without significantly increasing the number of optic lobe and larval eye cells. By contrast, later heat pulses (after 5 hours) lead to larval eye/optic lobe hyperplasia but no concomitant cyclops phenotype (Chang, 2001).
The dachshund (dac) gene encodes a novel nuclear protein that is required for normal eye development in Drosophila. In the absence of dachshund function, flies develop either without any eyes, or with severely reduced eyes. Targeted expression of dachshund is sufficient to direct ectopic retinal development in a variety of tissues, including the adult head, thorax and legs. This result is similar to that observed with the highly conserved Drosophila gene eyeless (ey), which can induce ectopic eye formation on all major appendages. dachshund and eyeless induce the expression of one another. dachshund is required for ectopic retinal development driven by eyeless misexpression. Dac protein is detected at the posterior margin of the eye disc prior to furrow initiation, and at positions both anterior and posterior to the furrow throughout furrow progression. Three independent results suggest that dac functions downstream of ey: (1) Misexpression of ey in the antennal, leg and wing imaginal discs is sufficient to induce ectopic dac expression in all discs. These results suggest that EY positively regulates dac expression. (2) Targeted expression of ey is
unable to induce ectopic eye formation in a dac mutant backfround. (3) ey is expressed in a mutant dac background, indicating that dac is not required for ey expression. These results suggest that the control of eye development requires the complex interaction of multiple genes, even at the very highest regulatory levels (Shen, 1997).
The eyeless, dachshund, and eyes absent genes encode conserved, nuclear proteins that are essential for eye development in Drosophila. Misexpression of eyeless or dachshund is also sufficient to induce the formation of ectopic compound eyes. Like ey and dac, targeted expression of eya alone is sufficient to induce ectopic eye formation. However, in contrast to ey, the penetrance of the ectopic eye phenotype induced by either dac or eya alone is incomplete and, when induced, such eyes are small. When dac expression was strongly induced in all imaginal discs, ectopic eye development was observed only on the anterior surface of the fly head ventral to the antenna, in just 56% (61/109) of animals examined. In contrast to the low penetrance of ectopic eye formation induced by dac or eya expressed alone, coexpression of dac and eya induces substantial ectopic eyes on the head, legs, wings, and dorsal thorax of 100% of animals examined. On the head, the cuticle between the normal eye field and antennae is transformed into retinal cells such that the normal retinal field is expanded. Large patches of pigment are induced on the dorsal surface of the femur and tibia of all legs, which are severely truncated. Ectopic eya alone can induce small patches of glass expression in the pouch area of the wing disc with 25% penetrance. In no case has ectopic Glass staining been observed in leg discs with either dac or eya alone. However, when dac and eya are coexpressed, ectopic Glass staining is induced with 100% penetrance along the ventral margin of the eye-antennal disc, the dorsal half of the leg disc along the anterior-posterior compartment (A/P) boundary, and along the A/P boundary of the dorsal wing disc. In each case, the sites of ectopic glass expression in discs correspond to the positions of ectopic retinal development observed in adults. Taken together, these data demonstrate that dac and eya show strong genetic synergy to induce ectopic retinal development in Drosophila (Chen, 1997).
While eya expression in the eye disc does not depend on dac function, dac expression is greatly reduced in an eya2 mutant background, demonstrating that dac expression requires eya activity. Similarly, eyeless (ey) induction of ectopic dac expression is greatly reduced in an eya2 mutant background. These results suggest that dac may function downstream of eya. Consistent with this interpretation, eya is unable to induce ectopic eye formation in a dac mutant background. eyeless misexpression is sufficient to induce eya, suggesting that eya may be required for ey function. Indeed, ectopic retinal development driven by targeted ey expression fails to occur in an eya2 mutant background. Induction of eya expression by ey does not depend on dac activity, consistent with the idea that eya functions downstream of ey but upstream of dac. However, these genes do not act in a simple, linear pathway; targeted expression of dac and eya strongly induce the expression of one another, and eya is required for ectopic eye induction by dac. Misexpression of dac or eya is also sufficient to induce ectopic ey expression in the antennal disc. These results suggest that multiple positive-feedback loops exist among these genes during normal eye development and raises the possibility that ey may be required for ectopic retinal induction by eya and dac. Indeed, ectopic eye formation driven by coexpression of dac and eya is completely blocked in an eyeless2 mutant background, indicating that induction of ey is essential. It is proposed that a conserved regulatory network, rather than a linear hierarchy, controls retinal specification and involves multiple protein complexes that function during distinct steps of eye development (Chen, 1997).
The fly eyes absent (eya) gene, essential for compound eye development in Drosophila, has been shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, has been defined: eya is found to be essential for eye formation by eyeless. Directing eyeless expression to discs that generate legs, wings and the antennal region of the head generates ectopic eyes in these regions. Eya is ectopically expressed in regions where eyeless directs ectopic eye formation. eya is essential for these ectopic eyes as ectopic eyes fail to form in eya mutants. eya can itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Directing ectopic eya expression to imaginal discs induces ectopic eyeless gene expression in the antennal region of the eye-antennal disc but fails to induce ectopic eyeless expression, even though ectopic ommatidia are formed and ectopic expression of Glass occurs. eya and eyeless together are more effective in eye formation than either gene alone: when expressed together, ectopic eyes are larger and form with higher penetrance than is the case with either eyeless or eya alone; expressed togther, eye formation occurs on the genitalia, a condition never observed in individuals with either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation (Bonini, 1997).
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).
The eyes absent gene is critical for normal eye development in Drosophila and is highly conserved to vertebrates. To define regions of the gene critical for eye function, the mutations in the four viable eya alleles have been defined. Two of these mutations are eye specific and undergo transvection with other mutations in the gene. These are deletion mutations that remove regulatory sequence critical for eye cell expression of the gene. Two other viable alleles cause a reduced eye phenotype and affect the function of the gene in additional tissues, such as the ocelli. These mutations are insertion mutations of different transposable elements within the 5' UTR of the transcript. Detailed analysis of one of these reveals that the transposable element has become subject to regulation by eye enhancer sequences of the eya gene, disrupting normal expression of Eya in the eye. More extended analysis of the deletion region in the eye-specific alleles indicatesthat the deleted region defines an enhancer that activates gene expression in eye progenitor cells. This enhancer is responsive to ectopic expression of the eyeless gene. This analysis has defined a critical regulatory region required for proper eye expression of the eya gene (Zimmerman, 2000).
Eyeless directly regulates rhodopsin 1 (rh1) expression in photoreceptor cells. rh1 is expressed specifically in photoreceptor cells R1 to R6. eyeless is expressed in both larval and adult terminally differentiated photoreceptor cells. The homeodomain of Eyeless binds to a palindromic homeodomain binding site P3/RCS1 in the rh1 proximal promoter, which is essential for rh1 expression. P3RCS1 can be replaced by binding sites specific for the Paired domain of Eyeless. P3RCS1 is conserved in the promoters of all Drosophila rhodopsin genes as well as in many opsin genes in vertebrates. Multimerized P3 sites in front of a basal promoter are able to drive the expression of a reporter gene in all photoreceptors. These results suggest that Pax-6/Ey directly regulates rh1 gene expression by binding to the conserved P3RCS1 element in the promoter (Sheng, 1997).
The development of the embryonic eye anlagen was examined making use of an eyeless-eye enhancer lacZ reporter. In wild-type embryos, the transgene drives beta-galactosidase expression in part of the morphologically distinct eye primordia. During larval stages, beta-galactosidase is continuously expressed in the eye discs and in parts of the brain. The position and number of cells that express this reporter in ey 2 mutant embryos is indistinguishable from wild-type embryos. It is concluded that the anlagen of the eye are formed in ey 2 mutant embryos. Therefore, defects in the first steps of eye development are not the major cause of the eyeless phenotype (Halder, 1998).
In contrast to the normal appearance of the eye anlagen in ey 2 embryos, the morphology of eye-antennal imaginal discs from late third instar ey 2 mutant larvae is highly abnormal, with the eye portion being strongly reduced. The antennal part is not affected. Staining for differentiating photoreceptors fails to show any evidence of ommatidial cluster formation in most ey 2 mutant eye discs. Previous work suggested that the ey 2 phenotype was a result of cell death in third instar eye discs. To assess cell death, eye discs were stained with the vital dye Acridine orange. A low level of cell death is normally observed in wild-type eye discs, mainly in the region just anterior to the furrow. In contrast, eye discs from third instar ey 2 larvae display massive cell death in the remainder of the eye discs. Eye discs with weaker phenotypes show ectopic cell death anterior to the furrow. This cell death phenotype is very similar to those observed in so 1 and eya 1 mutants (Halder, 1998).
To gain insight into the epistatic relationships among ey, so and eya, 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).
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).
Genes involved in eye development are highly conserved between vertebrates and Drosophila. Given the complex genetic network controlling early eye development, identification of regulatory sequences controlling gene expression will provide valuable insights toward understanding central events of early eye specification. The focus of this study is the defining of regulatory elements critical for Drosophila eyes absent expression. Although eya has a complex expression pattern during development, analysis of eye-specific mutations in the gene reveal a region selectively deleted in the eye-specific alleles. Detailed analysis has been performed of a small 322 bp region immediately upstream of transcriptional start that is deleted in the eye-specific eya2 allele. This analysis shows that this region can direct early eya gene expression in a pattern consistent with that of normal eya in eye progenitor cells. Functional studies indicate that this element will restore appropriate eya transcript expression to rescue the eye-specific allele. Regulation of this element during eye specification has been examined, both in normal eye development and in ectopic eye formation. These studies demonstrate that the element is activated upon ectopic expression of the eye specification genes eyeless and dachshund, but does not respond to ectopic expression of eya or sine oculis. The differential regulation of this element by genes involved during early retinal formation reveals new aspects of the genetic hierarchy of eye development (Bui, 2000).
The eya enhancer is expressed in ey, so, and dac mutant eye discs in a pattern consistent with previous studies of Eya protein expression during normal eye development. Normally, eya expression is dependent upon ey activity, partially dependent upon so activity, and independent of dac activity. Regulation during ectopic eye formation was addressed in order to define genes that control the expression of this eya enhancer region and to observe differential activation of the eya enhancer. Activity of the enhancer was detected upon ey- and dac-induced eye formation, as anticipated by previous studies. However, enhancer activation is not apparent upon ectopic eya or so gene expression or the combination of eya and so together. Thus, this eya enhancer appears to be selectively activated during ectopic eye formation, indicating a molecular distinction in how ey and dac genes induce ectopic retinal tissue compared to induction by the eya and so genes, at least with respect to regulation revealed by this element (Bui, 2000).
The regulation of this defined eye enhancer for eya suggests that eya and so function distinctively, at least in part, from dac and ey in ectopic eye formation. Whereas ey and dac either directly activate or feedback to activate eya expression, eya and so do not participate in regulatory loops to the level of activation of eya gene expression as defined by the eya eye enhancer (Bui, 2000).
eya can also synergize with dac in ectopic eye development, and physically interact with the Dac protein. However, the loss-of-function phenotype of dac in the eye is not identical to that of eya and so. These studies also suggest that dac is not acting the same way as eya with respect the eya enhancer: dac strongly activates expression, but eya does not. Based on observations from expression studies, dac has previously been placed downstream of eya. However, Dac is reduced, but not missing from eya mutant eye discs. The reduced expression may reflect massive loss of eye progenitor cells in eya mutant eye discs; alternatively, or in addition, there may be a partial dependence of Dac expression upon eya gene function. Thus, Dac may indeed be involved normally in aspects of eya gene expression. Previous studies showing Eya expression on ectopic eye formation are confounded by the fact that Eya is expressed both prior to and after the appearance of the furrow, but this expression is likely to be under the control of different regulatory elements. The element defined here presents a probe for at least some aspects of the early regulation of eya gene expression. The functional requirement by eya for ey and dac activity (and vice versa) in ectopic eye formation may reflect concurrent roles or other, later roles of these genes in eye formation. ey clearly has multiple roles at distinct times in eye development, such as regulation of genes important for late events of photoreceptor cell differentiation, in addition to the early function stressed here (Bui, 2000).
With respect to eya enhancer activation, ey and dac may directly bind to the eya eye enhancer or the regulation may be indirect through additional, yet-to-be defined genes. It is suggested the regulation may not be direct, at least for Ey, as Ey binding sites are not clearly apparent within the element. Whether Dac protein directly binds to DNA has yet to be determined, but it likely interacts with known transcriptional regulators in addition to interacting with Eya. Yeast one-hybrid experiments have also failed to support direct activation of the eya enhancer by Dac or Ey (as well as confirmed lack of activation by Eya and So). These studies provide a framework from which to define additional molecular genetic controls on early retinal specification. Recent studies showing that the fundamentals of ey/Pax-6 regulation can cross species boundaries suggests that not only are elements of the genetic pathway controlling eye development conserved in vertebrates, but fundamental aspects of the regulatory mechanisms may also be conserved. Given that vertebrate Eya homologs display functional rescue of Drosophila eya mutants, key regulatory aspects of eya gene expression, in addition to the function of the protein, may also be conserved. Eya is a critical gene of eye formation, with complex regulation of expression as shown here, as well as complex protein interactions, and multiple downstream targets. This eye enhancer controlling early eya expression provides a molecular genetic tool to help dissect additional regulatory events of eye specification that are involved in the conserved pathways of eye formation (Bui, 2000).
The Drosophila rhodopsin genes (rhs) represent a unique family of highly regulated cell-specific genes, where each member has its own expression pattern in the visual system. Extensive analysis of the rhs has revealed several functional elements that are involved in cell-specificity. The functional role of the RCSI/P3 site, which is found in the proximal promoter of all Drosophila rh genes, was investigated. This sequence is remarkably conserved in evolution and is located 15-30 bp upstream of the TATA box. In the context of the rh1 promoter, this element is recognized in vivo by a Pax6 protein, the master regulator of eye development. Thus, rh regulation might represent the ancestral function of Pax6. The role of the RCSI/P3 sequence in the other rh genes has been investigated and they have been found to also mediate Pax6 function. The potential impact of the various RCSI/P3 sequences on the precise cell-specific expression of rh genes was also investigated. Even though all RCSI/P3 sequences bind Pax6, they are clearly distinct in various rh promoters and these differences are conserved throughout evolution: RCSI/P3 appears to participate in the fine-tuning of cell-specificity. Pax6 or a related Pax protein may be involved in the regulation of olfactory genes. Therefore, in addition to performing a global photoreceptor-specific function, RCSI also appears to mediate the combined action of Pax6 and other factors and to contribute to rh regulation in subsets of photoreceptors (Papatsenko, 2001).
Based on the fact that all rhs are only expressed in photoreceptors (PRs), one might have expected to find common sequence elements among these related genes. Initial attempts to define common features of rh promoters by sequence comparison within a range of rh minimal promoters (from ~ -500 to +100) have resulted in the discovery of only two elements common to all genes: the TATA-box and the so-called RCSI sequences (Rhodopsin Conserved Sequence I). Both of these elements are necessary for transcription of all rh promoters; their mutation causes a total loss of activity. The homology between RCSI and the palindromic Paired-class homeodomain binding sites (P3 subtype) has suggested a functional link between RCSI and the critical regulator of eye development, Pax6. The direct interaction between what is believed to be a dimer of the Pax6 homeodomain (HD) and RCSI/P3 has been demonstrated genetically and molecularly for the rh1 gene (Papatsenko, 2001 and references therein).
Interestingly there are no sequences other than RCSI that are common to all rh promoters. At best, a few similar and/or evolutionary conserved regions can be detected, but only among certain subsets of the rh promoters. Moreover, in some cases, such as the rh3 and rh4 promoters, virtually no common sequences outside of the TATA box and RCSI can be observed, despite the fact that these two genes are both expressed in R7 PRs (rh3 in R7 pale, rh4 in R7 yellow). Based on these observations, a formal principle for the organization of rh promoters has been proposed: the proximal part, including RCSI, confers photoreceptor specificity. Distal upstream sequences, together with the proximal part, direct photoreceptor subtype specific expression. Although the separation of these two functions might not be absolute, it helps define the different functional units of rh promoters and facilitates their further dissection (Papatsenko, 2001).
The RCSI sequence, which contains a potential Pax6 HD recognition motif, was replaced by Pax6 PD binding sites in the rh3, rh5 and rh6 promoters. Despite the presence of ectopic expression in some cases, all target promoters maintained their original expression. The Pax6 PD and HD sites share no common sequence, but Pax6 binds both with high affinity. In many transgenic lines, the Pax6 PD binding sites have provided correct qualitative activity to replace the original RCSI HD elements. Therefore it is concluded that the RCSI site in all rh promoters contains a functional Pax6 homeodomain binding site. This site is necessary for the activation of all rh genes in the compound eye and thus, the Pax6 protein can be considered as a general activator that does not play a significant role in the extreme photoreceptor subtype specificity of the rh promoters. However, despite the fact that most modified promoters retain expression in their original location, different side effects, such as expansion into other classes of PRs, are also observed. This suggests that the presence of a functional Pax6 site alone is not sufficient to replace the RCSI sequence, since it is not sufficient for perfect promoter regulation (Papatsenko, 2001).
RCSI swap experiments among rh3, rh5 and rh6 were performed in order to investigate whether each RCSI sequence contains not only a general Pax6 site, but also other photoreceptor-specific information. This possibility is supported by the distinct structure of the RCSI sequence in each rh promoters. Conserved variations among families of RCSI sites might contribute to rh promoter specificity by either recruiting heterodimers of Pax6 with other Prd-class HD transcription factors, or some homeodomain proteins, such as K50 homeoproteins, to the RCSI element rh (Papatsenko, 2001).
The main result of the swap experiments was essentially the same as for the PD substitution: all modified promoters correctly maintain expression in their original location, but in several cases, ectopic expression patterns are observed. Another consequence of the RCSI swaps is considerable change in expression levels. This may be an indication of different affinity of the Pax6 site within different RCSI sequences. Based on the analysis of several lines (at least ten) for each construct, the strength of the different RCSI can be ordered: rh6 > rh3 > rh5. These swap and replacement tests suggest that Pax6 plays a similar activator role for the regulation of all rh promoters (Papatsenko, 2001).
The presence of similar Pax6 sites in all RCSI sites does not explain, however, the ectopic expression observed in several instances. One simple explanation might be the different strengths of Pax6 sites. Incorrect affinity of the main regulatory site might lead to such unexpected patterns. In particular, a stronger site might override other regulatory interactions. For instance, it is possible that Pax6 binding sites of strictly defined strength are required to provide the correct pattern of a given rh promoter. A second explanation for the ectopic expression is the presence of other regulatory motifs within RCSI, together with Pax6 sites. Disruption of these binding sequences in the replacements may explain the observed disruption of promoter specificity. The RCSI sequences might in fact be composite elements that differ in each rh promoter (Papatsenko, 2001).
Although Pax6 appears necessary for the activation of all rh genes through the RCSI, the results suggest that this interaction may not be sufficient for perfect rh expression. RCSI might represent a composite element that interacts with several regulatory molecules. For instance, a Pax6 homodimer might bind to its palindromic site, unless a heterodimer of Pax6 and another Prd-class HD protein bind to this site. Finally, a regulatory protein other than a homeoprotein might bind to RCSI, thus providing a third factor that either forms a complex with, or might compete with Prd-class homeoproteins (including Pax6). Among the potential candidates for the heterodimer model, the two Pax6 genes, ey and twin of eyeless (toy) might act together. It is not known which Pax6 gene, toy or ey acts on RCSI in vivo. Both Ey and Toy have similar affinity to the Pax6 HD sites (RCSI), and thus, they could be both involved in regulating rh genes through binding to RCSI as homo- or hetero-dimers. Although ey expression is turned off after the furrow in the third instar larvae, it is re-expressed during larval life. Currently there are no reports describing expression of toy in the adult. Since both Ey and Toy have a PD, the PD-replacement experiments could reflect the function of either gene. However, it has been reported that the PD of Ey binds poorly to DNA as compared to Toy, due to a mutation at a critical residue of the PD, suggesting that Toy is the protein binding to the PD replacement promoter. It must be noted, however, that the Toy binding sites found in the ey promoter are quite different from the Pax6 PD sites defined in vitro and the synthetic PD site used in this study (Papatsenko, 2001).
RCSI might also represent a site for a heterodimer of Pax6 (Toy or Ey) with another homeoprotein from the Prd-class. Binding of such heterodimers to RCSI might lead to different transcriptional outcomes: repression versus activation. This model is supported by the presence of K50 motif in the rh3 and rh6 RCSI sequences. Both RCSI sequences contain the motif CTAATCC that perfectly matches the binding consensus for a K50 subclass of homeoproteins, such as Gsc and Otd, which are known as repressors. In this case, a Pax6-K50 heterodimer might form a repressor complex on RCSI. There are also examples where K50 HD sites overlap other recognition sequences. For instance, the even-skipped stripe2 enhancer has K50 Bicoid activator binding sites (CTAATCC) overlapping binding sites for the Kruppel repressor (Papatsenko, 2001).
The swap experiments show that the Pax6 PD sites can fully replace the Pax6 HD sites only in one case out of three (rh5). In a few instances, replacement of the RCSI of one gene by another does maintain a correct expression pattern. However, it is clear that, in most cases, substitution of a native RCSI sequence by a generic Pax6 site is not fully sufficient to maintain exclusive expression. Therefore, a proper, specific structure for RCSI is required to achieve non-overlapping, specific patterns of expression. These facts, taken together with the presence of other known motifs (K50) within some RCSI sites, suggest a complex role for this potential composite element (Papatsenko, 2001).
In all the experiments, the initial specificity of the promoters was always preserved since ectopic expression was observed in addition to the expected pattern. This effect is more likely to be caused by disruption of existing repressor elements within RCSI rather than by creating new activator sites. Therefore some repressor sites might be present within at least some RCSI elements. For instance, rh5 and rh6 can be expressed in R7 cells upon changing their RCSI sequence; rh3 can also be expressed in all R7 when its RCSI is changed. Normally, rh5 and rh6 are expressed in two non-overlapping subsets of R8 PR's. However, in several cases, expression of these genes is observed in R7 cells. In one case, ectopic expression in R7 was obtained as the result of swapping the RCSI sequence of rh5 with that of rh6. In the other case, it was the result of rh6 RCSI replacement by Pax6 PD sites. It is possible that the rh5 and rh6 promoters both have an intrinsic potential to be expressed in both R7 and R8, but are selectively repressed by another factor in R7 cells. The putative R7 repressor factor might bind in the proximity or within the RCSI sequence, and its binding site might be affected by replacement of the RCSI sequence. Through extensive sequence analysis, a motif common to rh5 and rh6, GNCTAAGNC has been identified that is within the highly conserved regions of these promoters. The best match to this motif (GGCTAAGAC) is located 67bp upstream of RCSI in rh5. However, a weak site overlaps the RCSI (aAtTAAGTC). At least two putative R7 repressor sites flank/overlap the rh6 RCSI with the strongest one downstream of the third HD core (GTCTAAGAC). Some of these potential repressor sites have been disrupted in these experiments, perhaps resulting in partial derepression of the R8 promoters in R7 cells. The function of the R7 repressor sequence in both rh5 and rh6 promoters is currently being tested (Papatsenko, 2001).
An apparent derepression effect is also observed when the rh3 RCSI is replaced by either Pax6 PD sites, or by a stronger RCSI. In this case, rh3, which is normally only expressed in the pale subset of R7, exhibits equal expression in all R7, becoming a pan-R7 promoter. Some of the wild type minimal rh3 lines are also expressed in a pan-R7 pattern, but in those cases, expression in R7 yellow (rh4-specific) is much weaker than in R7 pale
(rh3-specific) and is restricted to the dorsal part of the eye. This derepression cannot be linked to the disruption of the K50 site in the rh3 RCSI since the replacement by the similar RCSI from rh6 does not restore the correct pattern. It is also difficult to explain this effect by a stronger Pax6 binding site that somehow overrides R7 yellow repression, since a swap with the weaker rh5 RCSI gives the same result. Another repressor binding sequence, different from Pax6 and K50 might overlap rh3 RCSI or be placed next to it. Sequence analysis of conserved sites within the proximity of rh3 RCSI identifies the sequence ATTCCG that is unique to rh3 RCSI and is highly conserved. In all the tests with rh3, at least two mutations were introduced into that sequence. Furthermore, mutation of this sequence to ATTtgG, also result in the same very strong pan-R7 pattern (Papatsenko, 2001).
Altogether, these experiments support a functional significance for this potential repressor element. The effects caused by changes in RCSI sequences lead to the general conclusion that regulation of Drosophila rh promoters occurs through the complex interplay of activation and repression. In particular, activation by Pax6 through the RCSI composite element might be regulated by several repressors, specific for different rh promoters. Together with the effect of upstream regulatory regions of the rh promoters, this leads to the formation of highly specific, exclusive expression patterns (Papatsenko, 2001).
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).
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 formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).
hern and fer genes are required for normal eye development and form eye tissue and activate ey when ectopically expressed. To study the role of the hern and fer genes in eye development, the eye phenotype was examined when either the hern or fer genes are inactivated by RNAi or are expressed ectopically. Expression of ds-hern or ds-fer RNA in the eye primordium with a GMR-GAL4 driver causes a slightly rough eye, with some bristles irregularly positioned. Curiously, the phenotype is not increased if the ds-hern and ds-fer RNAs are induced in the same fly. Misexpression experiments also suggest that both hern and fer are involved in eye development. Thus, the expression of either hern or fer with different GAL4 drivers causes the appearance of ectopic eye tissue in the third antennal segment or rostral membrane. These transformations are accompanied by the ectopic expression of ey, although this effect may also indicate the maintenance of a previous ey expression. Conversely, the misexpression of ey activates the hern and fer genes ectopically. Both hern and fer also activate embryonic lethal abnormal vision (elav), a marker of neuronal differentiation, when ectopically expressed. The analysis of clones expressing the fer gene in the leg, eye-antennal, or wing discs shows that elav activation is strictly nonautonomous, and only occurs in some cells adjacent to some of these clones (Suzanne, 2003).
The formation of the morphogenetic furrow in the eye is limited laterally by wg signaling. hern and fer expression within the eye primordium includes the more lateral wg-expressing regions. Interestingly, both hern and fer activate wg transcription when ectopically expressed. In ptc-GAL4/UAS-hern or dpp-GAL4/UAS-fer flies, the wings show several alterations, including the appearance of marginal bristles in the middle of the wing blade. This phenotype is characteristic of ectopic wg signaling, and in fact, wg is ectopically expressed in the wing discs of these larvae. Clones expressing the fer genes in the eye-antenna, leg, or wing discs also show induction of wg, mostly within but also outside the clone. The elav gene is also induced nonautonomously by these clones. Cells ectopically expressing elav do not coincide with those expressing wg and this reproduces the wild-type situation in the eye (Suzanne, 2003).
Signaling pathways can modify the activity of selector genes and are needed for proper organ formation. N signaling, for instance, is needed for eye formation and can activate ey when ectopically activated. Moreover, N has been implicated in the decision of making eye or antenna, directing eye development, and suppressing antenna formation. Therefore, whether N signaling could alter the ey and elav expression induced by the Tintin genes was examined. The coexpression of the hern gene and a dominant negative form of the Notch receptor substantially reduces ey and eliminates elav ectopic signals. Accordingly, no ectopic eyes are formed in this genetic combination. This indicates that the effect of hern on ey expression and eye formation requires N signaling (Suzanne, 2003).
Several eye-specifying genes have been identified, and they fulfill two conditions: they are required to make the eye and they can form ectopic eyes when expressed in different parts of the body. The hern and fer genes probably form part of this network of 'eye-specification' genes: (1) they are expressed in the eye primordium, with higher levels of expression anterior to the morphogenetic furrow; (2) they activate ey and elav and make ectopic eyes when expressed ectopically; (3) ey also activates the hern and fer genes when ectopically expressed. hern and fer genes have also been identified as downstream of ey in eye ectopic formation (Michaut, 2003). However, the inactivation of both hern and fer genes by RNAi with the GAL4 driver does not grossly affect eye development, as do mutants in the eye-specification genes. The nonautonomous induction of elav when hern or fer are ectopically expressed reproduces the wild-type situation, in which high levels of hern and fer are observed adjacent to the differentiating, elav-expressing, photoreceptor cells. Another similarity of hern and fer with some of the 'eye-specification'genes is that ectopic eye tissue is obtained in the antennae. The eye-specification genes eya and dac also form eyes predominantly, when ectopically expressed, in this same position. This is perhaps due to ey being expressed in the antennal primordium in late embryos, thus providing a favorable genetic context for eye formation. In accordance, when either the hern or the fer gene is ectopically expressed, ectopic ey expression is detected only in the antennal disc. Eyes are also obtained in the rostral membrane when ectopically expressing the fer gene. This may be due to the absence of hth, since high levels of either hern or fer repress hth and removal of this gene in the rostral membrane forms ectopic eyes (Suzanne, 2003).
The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).
Transformations are observed of third antennal segment, where hern and fer are normally transcribed, to eye tissue, in Dll-GAL4/UAS-hern or dpp-Gal4/UAS-fer flies. This suggests that the levels of Hern and Fer products may be important in inducing or maintaining ey expression and distinguishing eye from antenna. Accordingly, when Hern or Fer products are increased in the antennal primordium, the expression of hth, an inhibitor of eye development, is eliminated. It is also noted that, in the wild-type eye-antennal discs, hern and fer show higher levels of expression in the eye primordium than in the antennal one, where these genes are coexpressed with hth. However, the amount of Tintin product is not the only factor in this distinction, since, for instance, in Dll-GAL4/UAS-hern eye-antennal discs, the area of ectopic ey transcription in the antenna is smaller than the area of hern overexpression. The activity of other genes will probably contribute to the formation of either eye or antenna. Thus, the ectopic expression of either hern or fer induces wg, an inhibitor of morphogenetic furrow formation, and this probably limits the places where the eye can develop (Suzanne, 2003).
Two recent models have been proposed to explain the specification of eye and antenna within the eye-antennal disc. Both models suggest that the activation of the N signaling pathway is a key element in this process. It has been suggested that N signaling activates both ey and Dll in the eye and antennal primordia; subsequently, ey represses Dll in the eye and perhaps the hth and extradenticle genes repress ey in the antenna. In this way, the exclusive expression of ey (in the eye) and Dll and hth (in the antenna) determine eye and antenna identity, respectively. It has been proposed that the N and Egfr signaling pathways (together with the hedgehog and wg genes) are instrumental in the decisions to make eye or antenna. N signaling has been proposed to promote eye development and prevents formation of the antenna, whereas Egfr signaling does the opposite. Ectopic expression of either hern or fer in the antenna induces ectopic eyes and activates ey and elav, but the coexpression of hern and an N dominant-negative protein does not result in ectopic eyes and almost eliminates ey and elav activation. This suggests that N function impinges on hern activity to form ectopic eyes. As in other cases, the combined activity of signaling pathways and selector genes determine the specification of different structures (Suzanne, 2003).
Pax-6 genes encode evolutionarily conserved transcription factors capable of activating the gene-expression program required to build an eye. When ectopically expressed in Drosophila imaginal discs, Pax-6 genes induce the eye formation on the corresponding appendages of the adult fly. Two different Drosophila full-genome DNA microarrays were used to compare gene expression in wild-type leg discs versus leg discs where eyeless, one of the two Drosophila Pax-6 genes, was ectopically expressed. These data were validated by analyzing the endogenous expression of selected genes in eye discs and identified 371 genes that are expressed in the eye imaginal discs and up-regulated when an eye morphogenetic field is ectopically induced in the leg discs. These genes mainly encode transcription factors involved in photoreceptor specification, signal transducers, cell adhesion molecules, and proteins involved in cell division. As expected, genes already known to act downstream of eyeless during eye development were identified, together with a group of genes that were not yet associated with eye formation (Michaut, 2003).
Transcripts corresponding to
5,600-6,100 genes have been detected in the eye discs. These genes may act
in eye development upstream or in parallel to ey, such as toy and optix, or may also be required
for leg disc development (Notch, Egfr, and
dpp). Therefore, despite their important role in eye
development, their transcription is not significantly up-regulated by
ectopic ey. The genes identified in this study are more likely to
be preferentially involved in retinal differentiation rather than being
required for general morphogenesis of imaginal discs. In agreement with
previous findings, the DNA microarrays detect an up-regulation of
eyes absent, so, and dachshund (dac), which encode evolutionarily conserved proteins functioning together with Pax-6 at the top of the eye developmental cascade. However, dac up-regulation
occurs at only 74% of confidence because it is already
highly expressed in leg imaginal discs in the absence of ey,
consistent with its role in leg development. Because leg imaginal
discs were used as the baseline for gene activity in this screen, genes
more specifically required for eye rather than leg development are
detected at a higher confidence level (Michaut, 2003).
Among the 38 transcription factors found to be both induced
during ectopic eye formation and expressed in the eye imaginal discs, 18 were already associated with eye development. They are
endogenously expressed in the vicinity of the morphogenetic furrow and known to be required during the first steps of photoreceptor differentiation. Among those, the E(spl)
transcripts m delta and m gamma are expressed in
the morphogenetic furrow. atonal is first broadly
expressed in cells ahead of the advancing furrow and then undergoes
successive refinements until it is expressed only in a single cell in
each ommatidium, the R8 cell, which is the first photoreceptor to
differentiate. rough controls the differentiation
of the R2 and R5 cells, which are subsequently differentiating, and the bunched gene is expressed in a
hedgehog-dependent stripe in the undifferentiated cells just anterior
to the morphogenetic furrow. The genes pebbled and
glass start to be expressed in the morphogenetic furrow, and their expression extends posteriorly in the differentiated
photoreceptors. ey also induces the ectopic
expression of lozenge, which is expressed in all
undifferentiated cells arising from the second wave of morphogenesis
that give rise to the R1/R6, R7, cone, and pigment cells (Michaut, 2003).
Among the other 20 transcription factors up-regulated during
ectopic eye formation, eight have been described as being involved in
other developmental processes. For instance, the roles of
lola, sequoia (seq), and
stich1 in embryonic nervous system development were
investigated on the basis on their loss-of-function phenotypes.
Similarly, loss-of-function mutations in the net gene, which
encodes the Drosophila homolog of MATH6, have been described
as affecting wing vein patterning. The endogenous
transcription of these four genes in eye imaginal discs and their
up-regulation during ectopic eye development suggest a
possible role during eye development. Moreover, the transcription of
these genes in the developing eye was independently confirmed by serial
analysis of gene expression (SAGE) transcript imaging of
purified cell populations from eye imaginal discs; SAGE tags
corresponding to lola, seq, stich1,
and net were indeed detected in cDNA libraries derived from
sorted populations of eye disc cells (Michaut, 2003).
The fruitless (fru) and ken
and barbie (ken) genes also encode transcription
factors that are both expressed in the eye discs and induced
by ey during ectopic eye development. Although
ken transcripts are present in the eye disc in several rows
of cells posterior to the morphogenetic furrow, no defects in
eye development or morphology are described for viable mutant alleles. One possibility is that these mutant
alleles do not affect ken function in the eye, similar to
the case of the fru alleles; fru-viable mutations cause anomalies in male courtship behavior and affect the sex-specific transcripts produced under the control of a distal promoter of the gene. fru is a multifunctional gene that encodes sex-nonspecific proteins in addition to the protein involved in male behavior. One or more of these proteins could be responsible for fru function in the eye (Michaut, 2003).
In addition to transcription factors, signal transducers represent an
important category of genes expressed in the eye discs and up-regulated
during ectopic eye formation. The expressions in eye discs of the Ras interactors Sur-8 and sprint as well as the APPL-interacting
protein 1 were confirmed. The specific expression of these
three genes in the area of the morphogenetic furrow and their
significant induction during ectopic eye development
argues in favor of a previously uncharacterized function during eye
development. Among the three Rac genes present in
Drosophila, only Rac2 is up-regulated during
ectopic eye formation. Rac GTPases act at various steps of
development by controlling changes in cell shape. These modifications of the actin cytoskeleton are mediated by actin-binding proteins. The data show that the Quail protein, which is involved in actin bundle assembly during oogenesis is also present in the eye discs posterior to the morphogenetic furrow and is up-regulated during ectopic eye development (Michaut, 2003).
Transcription of a number of genes required at various steps of cell division is up-regulated during ectopic eye formation; twins encodes the regulatory subunit of protein phosphatase type 2A involved in regulation of mitosis and is expressed in imaginal discs. greatwall encodes a putative protein kinase required for chromosome condensation and mitotic progression, and skeletor encodes a chromosomal protein relocalizing during mitosis, which was postulated to constitute a matrix for assembly of the microtubule-based spindle during prophase. skeletor is expressed in the eye disc in a discrete row of cells posterior to the morphogenetic furrow; this row of cells could correspond to the cells undergoing the second wave of mitosis (Michaut, 2003).
The chit gene encodes a chitinase-related imaginal
disc growth factor synthesized by the fat body and involved in the
control of imaginal disc growth. chit
is also transcribed in leg and eye imaginal discs and its
transcription is increased during ectopic eye formation,
indicating an autonomous role of chit in imaginal disc
development and more specifically in eye differentiation. This finding
is in perfect agreement with the results of a microarray analysis of
genes differentially expressed in the various imaginal discs, where chit expression was found to be 2-fold higher in eye-antennal than in
wing discs (Michaut, 2003).
More than half of the 371 ey-induced genes identified
in this study are uncharacterized. No molecular function could be
assigned to 117 of them, such as SP1173 (FBgn0035710), for which no
homolog nor any functional domain could be identified clearly.
Interestingly, SP1173 transcripts are present in two distinct regions
of the eye discs: in a band of cells located in the area of the
morphogenetic furrow and at the posterior edge of the disc.
Transcription of three previously uncharacterized genes potentially
encoding cell adhesion molecules is also up-regulated during ectopic
eye formation: CG13532, BcDNA:gh11973, and CG9134 are expressed in the
area of the morphogenetic furrow. CG12605 encodes a putative
transcription factor similar to the pan-neural gene scratch
and is expressed posterior to the morphogenetic furrow, where neuronal
differentiation occurs. CG11849 and CG13651 encode homolog
proteins containing a N-terminal pipsqueak-DNA binding domain.
Both are ectopically induced by ey in the leg imaginal discs
and display almost identical expression patterns in the eye discs, in
nondifferentiated cells anterior to the morphogenetic furrow.
These genes encode putative transcription factors that may represent
previously uncharacterized, important regulators of eye development (Michaut, 2003).
BcDNA:gh11415 encodes the homolog of the
evolutionarily conserved cell fate-determining protein mab-21
identified in the nematode, zebrafish mouse and human. The mouse
mab-21 homolog participates in cerebellar, midbrain, and eye
development. In midgestation embryogenesis, it is expressed at its
highest levels in the rhombencephalon, cerebellum, midbrain, and
prospective neural retina. The human mab-21 homolog, CAGR1, was
detected originally in a retinal cDNA library; it is expressed in
several tissues, most prominently in the cerebellum.
BcDNA:gh11415 expression anterior to the
morphogenetic furrow in Drosophila eye imaginal discs and its ectopic induction by ey are consistent with an
evolutionarily conserved role of mab-21 in eye development (Michaut, 2003).
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 yields 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 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; full text of article).
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 differ