sine oculis



Drosophila eye development is under the control of early eye specifying genes including eyeless (ey), twin of eyeless (toy), eyes absent (eya), dachshund (dac) and sine oculis (so). They are all conserved between vertebrates and insects and they interact in a combinatorial and hierarchical network to regulate each other's expression. so has been shown to be directly regulated by ey through an eye-specific enhancer (so10). The regulation of this element has been studied; both Drosophila Pax6 proteins, namely Ey and Toy, bind and positively regulate so10 expression through different binding sites. By targeted mutagenesis experiments, these Ey and Toy binding sites were disrupted and their functional involvement in the so10 enhancer expression in the eye progenitor cells was studied. A differential requirement has been shown for the Ey and Toy binding sites in activating so10 during the different stages of eye development. Additionally, in a rescue experiment performed in the so1 mutant, the Ey and Toy binding sites were shown to be required for compound eye and ocellus development, respectively. Altogether, these results suggest a differential requirement for Ey and Toy to specify the development of the two types of adult visual systems, namely the compound eye and the ocellus (Punzo, 2002).

All animals analyzed so far, ranging from flatworms to mammals, have a Pax6 gene that is universally required for eye specification, according to the current state of knowledge. In contrast to vertebrates, where generally a single Pax6 gene gives rise to several differentially spliced transcripts, Drosophila and other holometabolous insects have two Pax6 genes, raising the question of functional redundancy. Gene duplication and subsequent divergence of developmental control genes is a major driving force in evolution, increasing the diversity and complexity of the organisms. A second mechanism for recruiting additional genes into a developmental pathway is enhancer fusion. The acquisition of new cis-regulatory elements represents an important mechanism for functional diversification. The findings reported in this study strongly support both of these hypotheses, since toy is able to rescue the eye development in an ey mutant when expressed in the ey domain. The finding that ey and toy exhibit different expression patterns during embryogenesis might account in part for their functional biological diversity. In the eye, both genes are co-expressed, except for the ocellar territory where only toy is expressed. In addition, it has been proposed that Toy and Ey diverged to regulate different sets of target genes because of a N14G mutation that changes the DNA binding specificity of the PD domain of ey. Indeed, using the so10 regulatory element it was found that Toy does not bind to the same sequences as Ey, but interestingly, Toy and Ey regulate the same target enhancer in different cells. The phenotypes obtained in rescue experiments using either the Ey/Toy or Toy binding site mutated enhancers, nicely parallel the phenotypes observed in ey mutants. The ey null mutant still has ocelli but lacks compound eyes. Interestingly escapers from the recently isolated toy mutant (toyG7.39) exhibit no eye reduction whereas the ocelli are partially missing. Therefore, removal of the common target gene of both Pax6 proteins in the eye (e.g. so1 mutant) consequently leads to a loss of both compound eyes and ocelli. Therefore, it is proposed that one of the developmental programs of toy is in part to specify ocellar development in addition to head formation, since toy mutants generated are characterized by pupal lethality, pharate adults lacking half of the head or the entire head capsule. Thus, it is proposed that the so gene is regulated by toy to specify the ocelli and by ey to specify the compound eyes during larval development (Punzo, 2002).

The analysis of ey and toy allows for the dissection of the evolutionary changes after the gene duplication event that happened during insect evolution. (1) The cis-regulatory regions of the two genes have diverged, leading to both temporal and spatial changes of expression; toy is expressed much earlier than ey during embryogenesis, whereas ey is not expressed in the ocellar region of the larval eye disc. (2) The protein coding regions of the two genes have diverged, most importantly in the paired domain where asparagine 14 (which is present in most Pax6 homologs), has been mutated in ey to glycine, which changes the DNA binding properties of the protein significantly. (3) The positive autocatalytic feedback loop found in vertebrates for their single Pax6 gene, has evolved into a heterocatalytic control loop in which toy transcriptionally activates ey by binding to the eye-specific enhancer of ey. (4) Both toy and ey cooperate in differentially regulating the so target gene, reflecting the fact that earlier in evolution so was regulated by a single Pax6 gene. These findings strongly support the hypothesis of intercalary evolution showing that the ey gene has been intercalated into the eye developmental pathway between toy and so. The observation that toy activates ey in the eye progenitor cells of the embryo, where neither so and eya are expressed, indicates that toy and ey are acting high up in the genetic hierarchy leading to eye development (Punzo, 2002).

In Drosophila, the sine oculis (so) gene is important for the development of the entire visual system, including Bolwig's organ, compound eyes and ocelli. Together with twin of eyeless, eyeless, eyes absent and dachshund, so belongs to a network of genes that by complex interactions initiate eye development. Although much is known about the genetic interactions of the genes belonging to this retinal determination network, only a few such regulatory interactions have been analysed down to the level of DNA-protein interactions. An eye/ocellus specific enhancer of the sine oculis gene has been identified that is directly regulated by eyeless and twin of eyeless. This regulatory element has been further characterized and a minimal enhancer fragment of so has been identified that sets up an autoregulatory feedback loop crucial for proper ocelli development. By systematic analysis of the DNA-binding specificity of so the most important nucleotides for this interaction have been identified. Using the emerging consensus sequence for SO-DNA binding a genome-wide search was performed and eyeless has been identified as well as the signalling gene hedgehog as putative targets of so. These results strengthen the general assumption that feedback loops among the genes of the retinal determination network are crucial for proper development of eyes and ocelli (Pauli, 2005).

In-vitro data on the autoregulatory element with the known so target sequence of lz and the AREC3/Six4-binding site, the consensus sequence GTAANYNGANAYC/G was identified as necessary for SO binding to DNA. This consensus sequence was taken as a basis for scanning the Drosophila genome for similar sites. In total, 1632 putative so targets emerged from this survey. Out of the affected genes several candidates are already known to be involved in eye development (Pauli, 2005).

so gene activity is crucial for proper development of the entire visual system of Drosophila, including the larval visual system (Bolwig's organ), the optic lobe, the compound eye and the ocellus. An eye-specific enhancer of so, so10, has been identified that is regulated by ey and toy. When used as a driver for so, so10 is sufficient to rescue only eye development of so1 mutant flies but not ocellus development. A fragment of 27 bp, soAE, found downstream of so10, is sufficient to rescue the entire mutant phenotype of so1 mutant flies when combined with so10. The So protein itself binds to soAE and, in cooperation with Eya, forms an autoregulatory feedback loop that is essential for ocellus development (Pauli, 2005).

Since So binds to its own enhancer and autoregulation cannot initiate expression of a gene, the initiation of so expression in the ocellar region must be triggered by other means. The following model is proposed. Initiation of so expression in early third instar eye discs is mediated by ey and toy throughout the eye disc, including the ocellar precursors. Later, after this first induction, so cooperatively with eya can maintain its own expression in the ocellar region by a positive autoregulatory feedback. Thus, the initiation of so expression is mediated by so10, whereas for the maintenance of so, soAE is required. This is supported by the observation that so10, which is activated by ey and toy, mediates expression in early third instar larvae all over the eye disc and only later gets restricted to the compound eye part (Pauli, 2005).

In this model the specificity of so expression for ocellar precursor cells is provided by the expression pattern of eya; Eya protein can be found only in the ocellar region itself, where it specifically interacts with So, and no Eya is present in the proximity of these cells. The importance of eya is further strengthened by the fact that eya4 mutants show an eyeless and ocelliless phenotype. Therefore, to elucidate the mechanisms that control gene expression specifically in ocellar precursor cells, additional studies on eya are required (Pauli, 2005).

Positioned at the top of the hierarchy of the retinal determination network, ey is a potent inducer of ectopic eyes and is able to directly induce so and eya. Like ey, so and eya are able to induce ectopic eyes but only when co-expressed; so alone fails to do so (Pauli, 2005).

To accomplish this induction, eya and so need to feed back on ey, obviously by binding to the eye-specific enhancer of ey. In an ectopic situation, the feedback of so/eya on ey is strong enough to induce ey for ectopic eye formation (Pauli, 2005).

The function of this feedback loop in normal eye development remains to be elucidated. so and eya are both expressed posterior to the furrow and are important for neuronal development. Nevertheless, ey is tuned down posterior to the MF. The activity of the so-binding site in the ey gene might, therefore, be suppressed by other factors or by so itself during cellular differentiation posterior to the furrow. Since co-expression of ey, so and eya is elevated only in a few cells in front of the MF and within the MF, a possible role for this feedback loop might be to boost ey expression in front of and within the furrow, which leads to a strengthening of so and eya expression in just a few cell rows (Pauli, 2005).

For proper eye development, a well-balanced expression level of the genes belonging to the retinal determination network is crucial. Loss-of-function mutations, as well as overexpression of the eye specification genes ey, eya, so or dac during eye development, impede proper determination of the organ and result in a reduction in eye size. Therefore, it is hypothesized that a feedback loop of so on ey is also important for the fine-tuning of ey expression during normal eye development. Due to its previously proposed ability to activate as well as to repress the expression of genes, so is a potent regulator in this context (Pauli, 2005).

decapentaplegic (dpp) signalling plays an important role in the complex regulatory network of eye development. In dpp mutant eye discs, so, eya and dac are not expressed, whereas dpp is able to initiate ectopic expression of so and dac when expressed at the anterior margin of the eye disc. Conversely, dpp expression is patchy in eye discs of eya and so loss-of-function mutants, suggesting that eya and so are required for either initiation or maintenance of dpp at the posterior disc margin before MF initiation (Pauli, 2005).

hh is required for dpp expression at the posterior margin before MF initiation, and dpp expression is induced by hh in the MF, supporting the assumption that dpp is downstream of hh signalling. Since dpp alone is not able to rescue posterior margin clones of hh, there have to be more eye-relevant target genes of hh signalling during third instar larval development. dpp in combination with eya can restore photoreceptor differentiation in posterior margin clones lacking smoothened (smo) expression (smo is a cell-autonomous receptor of hh signalling). This shows that dpp, in combination with eya, is able to bypass the requirement of hh during eye development. Taken together, it is evident that hh is necessary for proper eya and dpp expression, both of which can induce so, and it contains two so target sites. It is therefore hypothesized that the transcriptional complex consisting of Eya and So, as with ey, might also feed back on hh in order to drive the furrow during late eye development. In this model the genetic cascade starts with hh, which induces dpp and eya, moves on to so and through the So/Eya complex feeds back to hh in order to maintain hh expression as a driving force of the MF (Pauli, 2005).

The impact of these so-binding sites in the hh enhancer on eye development becomes evident from the fact that hh1 (bar-3) mutant flies have smaller eyes. The severity of the hh1 mutant phenotype is probably diminished by an additional putative So-binding site that resides outside the area covered by the hh1 deletion. If functional, this region (5' to the hh1 deletion) might mediate a residual hh-expression that overcomes the loss of the other sites to some extent. Another possible explanation for the rather weak hh1 phenotype might be that the feedback of so on hh is not crucial for MF initiation but still might be of importance for the well-balanced expression of hh during MF propagation (Pauli, 2005).

so belongs to the Six gene family. All Six proteins are characterized by a Six domain and a Six-type homeodomain, both of which are essential for specific DNA binding and protein-protein interaction. Based on the amino acid sequence of their homeodomain and Six domain, the Six genes were divided into three subgroups. Each of the three Drosophila homologues can be assigned to one of these subgroups: so is mostly related to Six1/2, optix to Six3/6 and DSix4 to Six4/5 (Pauli, 2005).

Promoter analyses of the mouse Six genes (Six1/2, Six4/5) revealed similar target sequence specificities for these mammalian counterparts of so. Six2, Six4/AREC3 and Six5 effectively bind to the same target sequence in a DNA fragment called ARE (Atpla1 regulatory element) that can be found in the Na,K-ATPase alpha1 subunit gene. Six1 and Six4 have been shown to bind to MEF3 sites in the myogenin and in the aldolase A muscle-specific (pM) promoters. Recently, mammalian Six4 has been shown to bind additionally to the transcriptional regulatory element X (TreX) within the muscle creatine kinase (MCK) enhancer (Pauli, 2005).

Comparison of all these sites confirms that the three nucleotides suggested to be the most important for So-DNA interaction are present and conserved within these motifs (nt. 1, 4 and 9 in the identifed binding site). In the case of the MEF3 site (which comprises seven nucleotides that include only two of the nucleotides important for So-DNA interaction), the original publications were examined to check if the third conserved nucleotide is also present, and in most of the cases its conservation has been verified. In fact, there is only one exception published in a study that describes two Six2 target sites (Pauli, 2005).

Nevertheless, by combining the vast majority of previous studies describing protein-DNA interaction of Six genes and this study of So-DNA interaction, it is inferred that So, Six1, Six2, Six4 and Six5 have very similar DNA-binding properties. In the case of so, it is proposed that the consensus sequence GTAANYNGANAY(C/G) marks a good starting point for the identification of additional targets of So, thereby helping to unravel the complex genetic interactions that orchestrate the development of the visual systems of Drosophila (Pauli, 2005).

Transcriptional Regulation

To gain insight into the epistatic relationships among eyeless, sine oculis and eyes absent, their expression patterns in eye discs were compared. Ey expression in the eye disc starts in the embryo and is later observed in the entire eye disc of late second and early third instars. During subsequent development, Ey expression is strong in the region anterior to the furrow and downregulated in differentiating cells. Very little, if any, expression posterior to the furrow or in the region of the developing ocelli in third instar eye discs is detected with polyclonal antibody or by in situ hybridisation. At the furrow, the expression patterns of Ey and Decapentaplegic (Dpp) abut each other, indicating that Ey expression is downregulated just before cells enter the furrow. Eya and So start to be expressed in eye discs later than Ey. In contrast to Ey, neither So nor Eya is expressed in the eye anlagen of stage-16 embryos. Expression of Eya and So in the eye disc starts in the late second and early third instar, respectively. At these stages, both genes are expressed in a gradient with the strongest expression at the posterior of the eye disc. Later, when the furrow moves across the eye disc, So and Eya are expressed in a graded fashion with strongest expression just anterior to the furrow. In this region the expression pattern of Ey overlaps with those of So and Eya. However, in the most anterior part of the eye disc only Ey is detected at high levels. Unlike Ey, So and Eya continue to be expressed posterior to the furrow. Both genes are also expressed in the region of the differentiating ocelli. In summary, Ey is expressed in the eye disc from embryonic stages onward, until cells enter the furrow and start to differentiate, while So and Eya start to be expressed later, and cells begin to express increasing levels of So and Eya as the furrow moves across the eye disc. These results are consistent with ey acting upstream of so and eya during eye disc development (Halder, 1998).

Gene expression was studied in ey 2, so 1 and eya 1 mutant eye discs. Genetic and molecular data indicate that the so 1 and eya 1 alleles are amorphic or severely hypomorphic in the developing eye. Because massive cell death is observed in late third instar eye discs of all three mutants, gene expression analysis at this stage is not possible. Expression patterns were therefore studied in early third instar eye discs. At this stage all three genes are expressed and cells in the so 1 and eya 1 mutant eye discs are still viable. Eye discs from ey 2 mutants, however, already show first signs of morphological abnormalities, indicating that ey function is required prior to this stage. In eye discs of so 1 and eya 1 mutants, Ey is expressed normally, indicating that the functions of so and eya are not required for Ey expression. However, neither SO nor Eya expression is observed in ey 2 mutant eye discs. This demonstrates that ey function is required for eye disc expression of So and Eya. In about half of the so 1 mutant eye discs weak Eya immunoreactivity is detected, suggesting that so may not be required for EYA expression. Expression of So is not seen in eya 1 mutant eye discs. However, because So and Eya are expressed in nearly identical patterns and because both genes are required for cell viability, these results are not conclusive. In summary, (1) ey acts earlier than and upstream of so and eya in the developing eye disc and (2) the functions of so and eya in the eye disc appear to be dispensable for ey expression (Halder, 1998).

To further investigate the epistatic relationships among ey, so and eya, gene expression was examined in the developing extra eyes induced by Gal4-directed ectopic expression of eyeless. In wild-type third instar larvae So and Eya are not expressed in the wing disc proper. However, in wing discs that develop ey-induced extra eyes, both genes are ectopically expressed in and surrounding the developing photoreceptor clusters. These results indicate that ey acts upstream of so and eya during extra eye development. In order to investigate the dynamics and the spatial restriction of the induction of so and eya expression, ey was ubiquitously expressed in a temporally controlled manner using a heat-inducible transgene. Expression of so and eya was monitored by assaying lacZ expression of so and eya enhancer-traps. Ubiquitous expression of ey was induced starting at 83 hours after egg laying during the mid third instar stage. At this time neither so nor eya are expressed in the wing disc proper and eya is not expressed in leg discs. Two heat shocks induce only weak ectopic expression of so and eya; do not induce extra eye formation in adult flies, and just barely affect their morphology. This suggests that higher or prolonged levels of Ey may be required to efficiently reprogram cells into the eye developmental pathway. Consistent with this, induction of extra eyes is efficient when larvae carrying the heat-inducible ey transgene are heat-shocked six times. Such animals readily induce ectopic expression of so and eya; nearly 100% of pharate adult flies developed extra eyes. Although Ey is expressed ubiquitously, induction of both genes is confined to regions close to the A/P boundary that do not express Wg but do express Dpp. Thus, Ey alone is not sufficient to induce so and eya, bearing in mind that only those cells that are close to a source of Dpp appear competent to express so and eya in response to Ey. The finding that ey positively regulates so and eya transcription raised the possibility that so and eya may be required downstream of ey for ectopic eye formation. Indeed, targeted expression of ey is unable to induce ectopic eye development in so 1 and eya 1 mutant backgrounds, although ectopic Ey protein is produced and functional as inferred from its deleterious effects. Consistent with the lack of ectopic eye production, no ectopic photoreceptors develop in wing discs of so 1 and eya 1 mutants following targeted expression of ey (Halder, 1998).

Advantage was taken of the ectopic induction of so and eya by Ey to find out whether Ey activates so and eya in parallel and independently of one another or whether induction of one gene depends upon the function of the other one. The cell death phenotypes observed in the eye discs of so 1 and eya 1 make such an analysis difficult in the eye discs. It was reasoned that by expressing ey ectopically those requirements for cell viability might be bypassed. However, in late third instar larvae, ectopic Ey expression in so 1 and eya 1 mutant backgrounds causes ectopic cell death in wing discs and results in strongly reduced and deformed adult structures. Apparently, Ey is able to completely reprogram wing cells into the eye developmental pathway, even if that leads to cell death, as is the case in so 1 and eya 1 mutants. Nevertheless, in early to mid third instar wing discs, Ey induces ectopic expression of eya in a so 1 mutant background and, conversely, so is induced by Ey in an eya 1 mutant background. Therefore, both genes appear to be independent targets of Ey. However, the ectopic expression is weaker than that induced in a wild-type background, suggesting that so and eya are required for efficient induction of each other's expression. In summary, these results show that Ey acts upstream of so and eya and requires their function during ectopic eye induction (Halder, 1998).

In addition to its function in the developing compound eye, so is required for the formation of the entire visual system, including the optic lobes of the brain and the larval photoreceptor organs known as Bolwig's organs. In blastoderm-stage embryos, so is expressed in a dorsal domain of the head region that gives rise to those structures. Whether this region also includes the primordia of the eye discs is unknown and no so transcripts are detected in the eye discs when they become morphologically discernible toward the end of embryogenesis. A second Pax-6 gene has been isolated from Drosophila, designated twin of eyeless (toy), which is expressed in the developing head from the blastoderm stage onward. In contrast, ey starts to be expressed at germ band extension. The early expression of toy overlaps so expression in the head and their epistatic relationship has been investigated. Cytologically, toy maps close to ey on the fourth chromosome. Since no mutations in toy have been identified thus far, advantage was taken of a compound fourth chromosome to generate nullo 4 embryos that lack both toy and ey functions. Such embryos express so at normal levels in the head, indicating that toy is not required for so expression in the embryonic head. Similarly, toy is expressed in an appropriate pattern in embryos homozygous for a null allele of so. Therefore, so and toy appear to act in parallel during the development of the embryonic head of Drosophila. Later in development, so null embryos express toy and ey in the eye anlagen indicating that so is not only dispensable for that expression but also for the initial formation of the eye anlagen (Halder, 1998).

decapentaplegic mediates the effects of hedgehog in tissue patterning by regulating the expression of tissue-specific genes. In the eye disc, the transcription factors eyeless, eyes absent, sine oculis and dachshund participate with these signaling molecules in a complex regulatory network that results in the initiation of eye development. Analysis of functional relationships in the early eye disc indicates that hh and dpp play no role in regulating ey, but are required for eya, so and dac expression. Ey is expressed throughout the eye portion of the wild-type eye disc during early larval stages, prior to MF initiation. Eya and Dac are expressed throughout the posterior half of the eye imaginal disc, with stronger expression at the posterior margin. Ey is expressed normally in homozygous Mad1-2 clones that touch the posterior margin and in clones that are positioned internally in the disc, indicating that Dpp signaling is not required for Ey expression prior to MF initiation. In contrast, neither Eya nor Dac is expressed in homozygous Mad1-2 clones that touch the margin of the eye disc. In addition, Eya and Dac are not expressed, or are expressed weakly, in internal clones that lie well anterior of the posterior margin. However, strong Eya and Dac expression is observed in internal clones that lie within a few cell diameters of the posterior margin. Like Eya and Dac protein, SO mRNA is expressed in the posterior region of the eye disc prior to MF initiation. Mad1-2 posterior margin clones fail to express so. These results suggest that dpp function is required to induce or maintain Eya, SO and Dac expression, but not Ey expression, at the posterior margin prior to MF initiation. This function is consistent with the pattern of DPP mRNA expression along the posterior and lateral margins at this stage of eye disc development. Whereas dpp is not necessary for Eya and Dac expression in internal, posterior regions of the early eye disc, it does play a role in regulating Eya and Dac expression in internal, anterior regions of the disc. Although DPP mRNA expression does not extend to the very center of the eye disc, it is expressed in a significant proportion of the interior of the disc. The possibility that dpp may regulate gene expression in more central regions may be attributed to the fact that it encodes a diffusible molecule (Curtiss, 2000).

Restoring expression of eya in loss-of-function dpp mutant backgrounds is sufficient to induce so and dac expression and to rescue eye development. Thus, once expressed, eya can carry out its functions in the absence of dpp. These experiments indicate that dpp functions downstream of or in parallel with ey, but upstream of eya, so and dac. Additional control is provided by a feedback loop that maintains expression of eya and so and includes dpp. The fact that exogenous overexpression of ey, eya, so and dac interferes with wild-type eye development demonstrates the importance of such a complicated mechanism for maintaining proper levels of these factors during early eye development. Whereas initiation of eye development fails in either Hh or Dpp signaling mutants, the subsequent progression of the morphogenetic furrow is only slowed down. However, clones that are simultaneously mutant for Hh and Dpp signaling components completely block furrow progression and eye differentiation, suggesting that Hh and Dpp serve partially redundant functions in this process. Interestingly, furrow-associated expression of eya, so and dac is not affected by double mutant tissue, suggesting that some other factor(s) regulates their expression during furrow progression (Curtiss, 2000).

The lack of eya, so and dac expression in Mad1-2 clones that lie at the margins of the eye disc prior to MF initiation reflects a role for dpp in controlling early eye gene expression at these stages of eye development. Evidence from several studies suggests that ey acts together with dpp at or near the top of the hierarchy: (1) ey expression is not regulated by dpp; (2) ey and dpp are both required for eya, so and dac expression prior to MF initiation; (3) ey is not capable of rescuing dppblk eye development or of inducing ectopic eyes in regions of imaginal discs in which dpp is not already expressed. These observations suggest that ey functions upstream of or in parallel with dpp. The possibility that ey is responsible for dpp expression, leading indirectly to eya, so and dac expression, is unlikely. Since ey cannot induce ectopic eyes without a source of dpp, it probably cannot induce dpp expression, at least not in the absence of factors that are specific to the eye disc. Moreover, Ey protein binds to the regulatory region of so, suggesting it is directly involved in so regulation. Thus, it is likely that ey and dpp cooperate to induce expression of the other early eye genes (Curtiss, 2000).

Such cooperation could achieve two ends. (1) ey is expressed throughout the eye disc and from embryonic stages of development through MF initiation. However, induction of eya, so and dac expression and MF initiation occurs approximately 48 hours later, around the time of the transition between second and third instars. Moreover, eya, so and dac are not expressed throughout the eye disc as ey is, but have stronger levels of expression around the margins than in other regions. The initiation of dpp expression at the posterior margin at approximately the same time suggests that it could be the spatiotemporal signal that sets the MF in motion. (2) dpp induces expression of tissue-specific genes as part of its role in patterning many diverse structures in Drosophila. An interaction with ey could be essential to ensuring that in the eye imaginal disc dpp initiates factors that are appropriate to eye development, such as eya, so and dac (Curtiss, 2000).

Bolwig's organ formation and atonal expression are controlled by the concerted function of hedgehog, eyes absent and sine oculis. Bolwig's organ primordium is first detected as a cluster of about 14 Atonal-positive cells at the posterior edge of the ocular segment in embryos and hence, atonal expression may define the region from which a few Atonal-positive founder cells (future primary photoreceptor cells) are generated by lateral specification. In Bolwig's organ development, neural differentiation precedes photoreceptor specification, since Elav, a neuron-specific antigen, whose expression is under the control of atonal, is expressed in virtually all early-Atonal-positive cells prior to the establishment of founder cells. Neither Atonal expression nor Bolwig's organ formation occurs in the absence of hedgehog, eyes absent or sine oculis activity. Genetic and histochemical analyses indicates that (1) the required Hedgehog signals derive from the ocular segment, (2) Eyes absent and Sine oculis act downstream of or in parallel with Hedgehog signaling and (3) the Hedgehog signaling pathway required for Bolwig's organ development is a new type and lacks Fused kinase and Cubitus interruptus as downstream components (Suzuki, 2000).

Prior to the establishment of Bolwig's organ founder cells, virtually all Bolwig's organ precursor (BOP) cells acquire neural fate. The earliest event of Bolwig's organ development may be ato expression at mid stage 10: this early ato expression defines the area of BOP. Early ato expression is regulated by the concerted action of Eya, So and Hh signals. During late stage 10 and early stage 11, Elav, a neuron-specific antigen, begins to be expressed in almost all BOP cells. This elav expression is likely to be regulated by Ato activity, since (1) BOP elav expression is reduced extensively in ato mutants and (2) the number of Elav-positive cells at stage 11 and Kr-positive Bolwig's organ neurons at stage 16 considerably increases upon ato misexpression. As with ato expression, eya, so and hh activity is essential for elav expression in BOP cells. In contrast to elav expression, ato expression is restricted to three founder cells at stage 12: this late ato expression disappears by the end of stage 12. Photoreceptor specification of putative founder cells may start during stage 11, since at late stage 11, 2-3 cells in a cluster start expressing Kr and/or Glass, which are specific markers for larval photoreceptors. Cells expressing Kr and/or Glass increase during stages 12-13 and all 12 photoreceptors express both Kr and Glass by stage 16. Similarly, a peripheral nervous system-specific signal recognized by mAb22C10 (see Futsch) appears in a few BOP cells at stage 12 and becomes recognizable in all Bolwig's neurons by stage 16. Late ato expression may also be essential for normal photoreceptor formation. In ato mutants, neither Kr-positive nor mAb22C10-positive cells can be seen in stage-16 future larval eyes (Suzuki, 2000).

Retinal cell fate determination in Drosophila is controlled by an interactive network of retinal determination (RD) genes, including eyeless, eyes absent, sine oculis and dachshund. The role of decapentaplegic in this pathway was investigated. During eye development, while eyeless transcription does not depend on dpp activity, the expression of eyes absent, sine oculis and dachshund are greatly reduced in a dpp mutant background. dpp signaling acts synergistically with, and at multiple levels within, the retinal determination network to induce eyes absent, sine oculis and dachshund expression and ectopic eye formation. These results suggest a mechanism by which a general patterning signal such as Decapentaplegic cooperates reiteratively with tissue-specific factors to determine distinct cell fates during development (Chen, 1999).

During ectopic photoreceptor determination there is a tight correlation between the location of ectopic eyes and the endogenous pattern of dpp expression. In particular, the dpp-GAL4 driver is the most efficient means of retinal induction by any of the RD genes: ubiquitous eyeless (ey) expression induces downstream genes only in the vicinity of the anteroposterior (AP) compartment boundary of discs where dpp is normally expressed. These results suggested that dpp signaling may be essential for the RD genes to specify retinal cell fates. dpp is normally expressed along the AP boundary of the larval wing disc. The GAL4 line 30A drives gene expression in a ring that surrounds the wing pouch, which will become the wing blade in the adult. The 30A ring pattern corresponds to tissue that will form the hinge of the adult wing and overlaps endogenous dpp at only two spots. When ey is misexpressed using 30A-GAL4, ectopic eye formation is induced only at two positions: dorsal and ventral to the pouch at the AP boundary. One explanation for this phenomenon is that dpp activity is essential for ey to induce ectopic eye development. Coexpression of dpp and ey is sufficient to expand the domain of ectopic retinal development induced by ey alone. To test whether dpp and ey act synergistically to induce RD genes, mRNA levels of ey, eya, so and dac were measured in a dpp loss-of-function background. ey is normally expressed throughout the entire eye disc prior to MF initiation and anterior to the furrow during MF progression. In dpp mutants, the eye-antennal disc is much smaller than in wild-type due to a proliferation defect, and MF initiation and photoreceptor development does not occur. Nevertheless, EY mRNA is still detectable in dpp mutant eye discs throughout second and third instar larval development. In contrast, although eya is still expressed in the ocellar region, almost no EYA, SO or DAC mRNA is detected in dpp mutant eye discs prepared from second or third instar larvae. These data indicate that dpp is not essential for ey expression but is required upstream of eya, so and dac in the eye disc (Chen, 1999).

If eya and dac are the primary downstream targets of dpp during eye development, then it should be possible to bypass the requirement for dpp and induce ectopic eye formation by overexpressing ey with eya or dac. While targeted expression of either eya or dac alone driven by 30A-GAL4 is unable to induce photoreceptor development, strong synergistic induction of ectopic eye formation is observed when ey is coexpressed with either dac or eya. Although there is clear synergy between ey and dac or eya, ectopic photoreceptor induction in both imaginal discs and adults is still limited to the vicinity of the AP boundary and the source of dpp signaling. Moreover, photoreceptor differentiation is still restricted to the vicinity of the AP boundary when ey, dac, eya and so are simultaneously induced by 30A-GAL4, indicating that dpp and ey must regulate other essential targets in this process (Chen, 1999).

It is possible that dpp signaling might cooperate directly and exclusively with ey. Alternatively, dpp could interact at multiple levels within this pathway. To distinguish these two models, a test was performed to see whether dpp functions synergistically with eya and so to regulate the expression of dac. No ectopic dac expression is induced by so alone: targeted expression of eya induces ectopic dac expression only at a single ventral spot on the AP boundary of the wing disc when driven by 30A-GAL4. Consistent with the idea that the Eya and So proteins function cooperatively as a complex, strong synergistic induction of dac is observed when eya and so are coexpressed. However, dac expression is still restricted mainly to places where endogenous dpp is present. In contrast, when dpp is coexpressed with eya, strong dac expression is induced all along the ventral-posterior pouch margin Moreover, ectopic Dac is detected around the entire circumference of the wing pouch as a result of dpp, eya and so coexpression. Since coexpression of dpp, eya and so is sufficient to induce dac expression in places where dpp and ey cannot, it is concluded that dpp interacts with the network at multiple levels to control the expression of retinal determination genes. Consistent with this interpretation, no induction of ey transcription could be detected in response to misexpression of dpp, eya and so with 30A-GAL4 (Chen, 1999).

Thus dpp signaling is reiteratively used to regulate gene expression within the retinal cell fate determination pathway in Drosophila. Specifically, dpp signaling enables ey to induce strong eya, so and dac expression in the posterior, but not anterior, wing disc compartment. In contrast, dpp functions synergistically with eya and so to activate the expression of dac in both compartments. This activation of dac expression by dpp, eya and so is unlikely to result from feedback induction of ey for two reasons: (1) targeted expression of ey and dpp is unable to induce dac in the anterior wing disc compartment, and (2) ectopic ey transcription is not detected in response to misexpression of dpp, eya and so driven by 30A-GAL4 in the wing disc. Thus, these data suggest that dpp signaling interacts with the retinal determination pathway at (at least) two levels to regulate RD gene expression. Interestingly, while targeted expression of dpp, eya and so with 30A-GAL4 is unable to induce ey expression or ectopic photoreceptor development in the wing disc, coexpression of eya and so using dpp-GAL4 is sufficient to induce ey expression and photoreceptor development in the antennal disc. These differences most likely reflect the unique transcriptional environments present in the specific portions of each imaginal disc tested in these assays (Chen, 1999).

The Pax-6 gene encodes a transcription factor with two DNA-binding domains, a paired domain and a homeodomain, and is expressed during eye morphogenesis and development of the nervous system. Pax-6 homologs have been isolated from a wide variety of organisms ranging from flatworms to humans. Since loss-of-function mutants in insects and mammals lead to an eyeless phenotype and Pax-6 orthologs from distantly related species are capable of inducing ectopic eyes in Drosophila, it has been proposed that Pax-6 is a universal master control gene for eye morphogenesis. To determine the extent of evolutionary conservation of the eye subordinate target genes of Pax-6, subordinate genes of Pax6 have been sought. Expression of two genes, sine oculis (so) and eyes absent (eya), is induced by eyeless (ey), the Pax-6 homolog of Drosophila. Evidence from ectopic expression studies in transgenic flies, from transcription activation studies in yeast, and from gel shift assays in vitro is presented supporting the notion that the EY protein activates transcription of sine oculis by direct interaction with an eye-specific enhancer in the long intron of the so gene. Sequences of the putative sites are at maximum 88% homologous (70% with the consensus PRD binding site sequence). The functional importance of the eye-specific enhancer of so has been demonstrated in vivo by means of the so1 mutant, which deletes a 1.3 kb region including the enhancer, but leaves the coding sequences intact. In so1 homozygous flies, ey is neither capable of inducing so transcription nor can it induce ectopic eyes. In contrast to ey, its paralog toy induces both ectopic so transcription and ectopic eyes in a so1 mutant background. This indicates that ey and toy regulate so by different mechanisms (Niimi, 1999).

teashirt was initially identified as a gene required for the specification of the trunk segments in Drosophila embryogenesis and encodes a transcription factor with zinc finger motifs. Targeted expression of teashirt in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a phenotype similar to that produced from targeted expression of eyeless, dachshund, and eyes absent. The expression of so and dac are induced in the antennal disc by the ectopic expression of tsh, suggesting that tsh may act upstream of these genes in eye development. Furthermore, teashirt and eyeless induce the expression of one another, suggesting that teashirt is part of the gene network that functions to specify eye identity (Pan, 1998).

However, these results do not prove that tsh does play a role in specifying the eye identity during normal development. To address this issue, an examination was carried out to see if tsh is expressed at the right time and the right place to have a role in specifying the eye identity. Indeed, TSH mRNA is expressed in the eye disc, with the strongest expression anterior to the morphogenetic furrow. This pattern of expression is similar to that of ey, a gene that is known to play an essential role in specifying eye identity. An examination was carried out to see if loss-of-function mutations of tsh affect eye development. Several weak loss-of-function tsh alleles were examined and no eye defects were found. X-ray-induced mitotic recombination was used to generate mutant clones of a null tsh allele. tsh mutant clones were recovered at a frequency similar to the wild-type control, and sections through the mutant clones revealed a normal ommatidial organization. These data suggest that tsh may play a redundant role during normal eye development, and the requirement for tsh may be masked by other factor(s) that play a role similar to tsh (Pan, 1998).

The function of the Dpp and Hh signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo has been analyzed. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain (Chang, 2001).

Dorsal epidermal and visual system fates, in particular those of the posterior optic lobe and larval eye, are not expressed in dpp loss of function. It is likely that these abnormalities are the result of changes in early head gene expression. This was followed in detail by assaying the expression of several regulatory genes known to be required for the normal development of the visual primordium, including otd, tll, so and eya in dpp-null mutants:

  1. otd is normally expressed in a wide domain that spans the dorsal midline and encompasses the entire dorsal head ectoderm. In normal development, its expression is turned off in the head midline (the head epidermis precursors) and in the part of the visual primordium forming the posterior optic lobe and larval eye. In dpp mutants, expression persists in the entire dorsal head ectoderm until stage 11. Expression then becomes patchy as many cells undergo apoptotic cell death (Chang, 2001).
  2. tll appears in the protocerebral ectoderm, including the head midline ectoderm. Only later does expression spread to cover part of the visual primordium. In embryos that lack Dpp, expression is expanded from the beginning to include the entire dorsal head. As for otd, expression also persists in the head midline ectoderm (Chang, 2001).
  3. so is expressed in a transverse stripe spanning the dorsal midline. This unpaired domain defines the eye field. Around gastrulation, so expression ceases in the dorsal midline and becomes restricted to the bilateral visual primordia. In addition to the visual system, so appears in the anlage of the stomatogastric nervous system (SNS) and head mesoderm. In a dpp-null fly, so is never expressed in the anlage of the visual system, although expression in the SNS and head mesoderm is unchanged (Chang, 2001).
  4. eya is normally expressed in a complex pattern that essentially consists of three domains located in the anlage of the SNS, the anterior protocerebrum and the anlage of the visual system. In dpp-null embryos, eya expression in the primordia of the visual system and SNS is absent from the beginning. The anterior protocerebral expression is narrowed (Chang, 2001).

The observed downregulation of head gap genes and early eye genes in the dorsal midline is an indirect effect of Dpp mediated by the Dpp target zerknüllt (zen). Previous studies have demonstrated that high levels of Dpp in the dorsal midline upregulate and focus the expression of zen in the amnioserosa and, further anteriorly, in the dorsomedial head epidermis. An RNA in situ probe revealed the expression of zen in the early eye field of a stage 5-7 embryo. Assaying the expression of head gap and early eye genes in a zen-null mutant background demonstrates that Zen acts as a repressor of these genes. Whereas in wild type, after an initial unpaired expression straddling the dorsal midline, tll, so and eya are turned off in the dorsal midline, they continue to be expressed in this domain in a zen mutant. At later stages, lack of zen results in a cyclops phenotype (Chang, 2001).

In the head region, highest levels of Dpp are required to promote mid dorsal fates (head epidermis, analogous to amnioserosa in the trunk). The activation of screw is involved in this process, similar to its role in the dorsomedial trunk. Intermediate Dpp levels promote dorsolateral fates (visual primordium). Low levels of Dpp are reached in the protocerebral neurectoderm and are permissive for the formation of protocerebral neuroblasts. Several of the regulatory genes expressed in the anterior brain and eye field may be direct targets of Dpp signaling. The findings show that so, eya and omb are activated by Dpp in the visual primordium. These regulatory genes initiate the fate of visual structures, in particular larval eye and outer optic lobe. It has recently been shown that eya and so are also targets of Dpp signaling in the eye imaginal disc (Chang, 2001).

The secondary restriction of so (and other genes with bilateral expression domains developing from unpaired domains, including tll and otd) is effected by the Dpp target zen in the dorsal midline. This homeobox gene is expressed as a response to peak levels of Dpp in the dorsal midline, including amnioserosa and, in the head of the embryo, in the dorsomedial head epidermis primordium. Loss of zen, similar to reduction of Dpp, results in the absence of amnioserosa and head epidermis, and a cyclops phenotype (Chang, 2001).

In view of these results, it is speculated that the interaction between Dpp and Hh is indirect and requires the function of so, eya and possibly other 'early eye genes' -- according to this model, Dpp activates so and eya in the eye field. Slightly later, expression of so and eya is lost dorsomedially, due to repression by Zen at this level. In a second step, the expression of Hh (which comes on later than Dpp) triggers larval eye fate in cells close to the Hh source. The response of a cell to Hh, that is, its expression of ato, depends on its previously expressing so and eya. Finally, Ptc inhibits the range of Hh action, similar to its alleged function in the trunk and imaginal discs (Chang, 2001).

A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:

  1. If the level of Dpp is reduced (in dpp null heterozygote, or dpp hypomorph), so and eya are stably expressed in the dorsal midline, since zen, which normally inhibits the early eye genes, is not expressed. As a result Hh can induce larval eye dorsomedially (Chang, 2001).
  2. In the cyclops phenotype that results from reduction of Dpp, the visual primordium develops as a double crested placode that spans the dorsal midline. In this placode, the posterior crest is formed by larval eye cells, in line with the tenet that Hh induces larval eye fate in the cells next to the Hh source (posteriorly). The anterior crest, which is further away from the Hh source, constitutes posterior optic lobe (Chang, 2001).
  3. In the cyclops phenotype induced by loss of Ptc or overexpression of Hh, larval eye cells are increased in number, compared with the Dpp reduction induced cyclops. At the same time, posterior optic lobe cells are reduced in number (Chang, 2001).

The Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).

In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin, or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless, suggests a possible function for Wingless in the regulation of the eye selector genes. The top of the genetic hierarchy involved in eye specification appears to be the Pax6 homolog, Eyeless. In the third instar eye disc the expression of Eyeless is restricted to the region anterior to the furrow and, despite the Wingless-induced inhibition of eye development, the expression of Eyeless in this region is not affected by axin- clones. This lack of an effect anterior to the furrow, despite the overgrowth and abnormal Distal-less expression in the same region, implies that misregulation of Eyeless is not the primary cause of the transformations caused by ectopic Wingless activity (Baonza, 2002).

Downstream of Eyeless (although feedback relationships makes the epistatic relationship complex) are other transcription factors required for eye specification, including Eyes absent, Sine oculis and Dachshund. A phenotype similar to axin- clones of excess proliferation and consequent overgrowth is caused by loss of Eyes absent and Sine oculis. Moreover, as in axin- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).

The expression patterns of Eyes absent, Sine oculis and Dachshund, in axin- and/or arm* mutant clones, were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin- M+ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin- M+ clones. Consistent with these results, in arm*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).

The conclusion that Wingless signaling negatively regulates the expression of Eyes absent, Dachshund and Sine oculis anterior to the furrow leads to the prediction that in normal development, domains of high Wingless activity in the anterior region of the eye disc will be associated with low expression of these genes. Previous work indicates that their expression is broadly non-overlapping, but to analyse this precisely, discs were double-labelled to detect the expression of Wingless and Eyes absent or Sine oculis throughout the third instar larval stage. The expression of these eye specification genes is precisely complementary to that of Wingless in the anterior lateral margins of the eye throughout the third instar. This is consistent with a role for Wingless signaling in initiating the borders between eye and other head structures. Note that in posterior lateral regions slight overlap is observed between the expression of Wingless and these genes; this is presumably analagous to the expression of eye specification genes seen in some posterior axin- clones, and confirms that in posterior regions of the eye disc, Wingless signaling is not incompatible with the expression of these genes (Baonza, 2002).

These results indicate that Wingless regulates the final size of the eye field of cells by controlling the expression of eyes absent, sine oculis and dachshund. The expression pattern of these genes in the anterior eye margin is complementary to the expression of Wingless throughout the third instar, indicating that in anterior regions, high activity of Wingless signaling corresponds to absence of these gene products. Moreover, ectopic activation of Wingless signaling represses their expression anterior to the furrow (where they act to specify the eye field) throughout eye development. Finally, the loss of Wingless signaling causes ectopic expression of Eyes absent and Dachshund (Baonza, 2002).

It is proposed that the initial expression of Eyes absent, Sine oculis and Dachshund is negatively regulated by Wingless signaling in the eye disc, and that this regulation initiates the border between the eye field and adjacent head cuticle. Attempts were made to define whether Wingless represses the eye specification genes independently or whether eyes absent is the primary target but the data confirms earlier reports of the complexity of the regulatory relationships between eyes absent, sine oculis and dachshund. The observation that Eyes absent is able partially to restore the expression of the other two genes but cannot rescue the overgrowth and differentiation phenotype of axin- clones has two possible explanations. Either Wingless represses eye development through at least one additional gene, or high level Wingless signaling blocks eye development later in the developmental program -- e.g., it is known to inhibit morphogenetic furrow initiation, even after its earlier effects are rescued by eyes absent expression (Baonza, 2002).

Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila

A general question in development is how do adjacent primordia adopt different developmental fates and stably maintain their distinct fates? In Drosophila, the adult eye and antenna originate from the embryonic eye-antenna primordium. These cells proliferate in the larval stage to form the eye-antenna disc. The eye or antenna differs at mid second instar with the restricted expression of Cut (Ct), a homeodomain transcriptional repressor, in the antenna disc and Eyeless (Ey), a Pax6 transcriptional activator, in the eye disc. This study shows that ey transcription in the antenna disc is repressed by two homeodomain proteins, Ct and Homothorax (Hth). Loss of Ct and Hth in the antenna disc resulted in ectopic eye development in the antenna. Conversely, the Ct and Hth expression in the eye disc was suppressed by the homeodomain transcription factor Sine oculis (So), a direct target of Ey. Loss of So in the eye disc caused ectopic antenna development in the eye. Therefore, the segregation of eye and antenna fates is stably maintained by mutual repression of the other pathway (Wang, 2012).

In l-L3 eye-antenna disc, although the expression domain of Ct/Hth and Ey/So are juxtaposed, so3 clones showed derepression of Ct and Hth only in the most posterior region (zone 4) but not in the more anterior regions behind MF (zones 2 and 3). Thus, there may be an additional mechanism to repress Ct and Hth expression. For Hth, the repression is by Dpp and Hh signaling in L3 eye disc. It has not been tested whether Ct is also repressed by Dpp and Hh (Wang, 2012).

For individual cells in the eye-antenna disc, the mutual repression provided a mechanism for a choice of bistable states, either eye or antenna fate. A bistable state can often be maintained by positive-feedback loop, in addition to mutual repression. Such a positive-feedback loop is known for the eye pathway, but has not been reported for the antenna pathway (Wang, 2012).

The mutual transcriptional repression mechanism is expected to work at the level of individual cells. Therefore, a salt-and-pepper mosaic pattern would be predicted unless there is additional patterning influence. The patterning gene dpp is expressed in the posterior margin of e-L2 eye disc, and is required for Eya expression at this stage. However, dpp is not required for the restricted expression of Ct and Ey. It is proposed that there is another patterning gene that biases the antenna disc to express Ct. Thus, the difference between eye and antenna primordia may be predetermined before the onset of Ct and Eya at e-L2 (Wang, 2012).

The subdivision of a developmental primordium into subprimordia with specific fates is a common requirement in development. For example, the mammalian ventral foregut endoderm differentiates into the adjacent liver and pancreas, and a bipotential population of foregut endoderm cells give rise to both liver and pancreas. The maintenance of such division by mutual antagonism has been reported before. For example, the division between the presumptive thalamus and prethalamus in Xenopus is due to the mutual repression by the Irx homeodomain proteins and the Fezf zinc-finger proteins. The boundary between optic cup and optic vesicle is maintained by mutual transcriptional repression between Pax6 and Pax2. The current findings provide a new example, with clear correlation, both temporal and causal, of gene expression changes and developmental fate specification (Wang, 2012).

The maxillary palp and ocelli are derived from specific regions in the eye-antenna disc. The maxillary palp fate does not become segregated from the rest of eye-antenna disc as late as late L3. The timing of ocelli fate decision is not clear. otd is required for ocelli development, and is the first marker for the ocellar region: it is ubiquitously expressed in the early L2 eye-antenna disc, and becomes restricted to the ocellar region in the eye disc in early L3. Thus, the palp and ocelli may be determined as subfields of the antenna disc and eye disc, respectively. This is consistent with the finding that hth>mi-ct+mi-hth (knocking down both ct and hth in their endogenous expression domain) resulted in the loss of palp, whereas so affected ocelli but not palp (Wang, 2012).

The results showed that Ct and Hth are repressed by So. Previous studies also found induction of Ct and Hth expression in so3 clones in a region far posterior to the MF in l-L3 eye disc. The fact that So represses Ct and Hth in two spatially and temporally distinct situations suggest that this is a conserved function of So (Wang, 2012).

Whether the repression of Ct and Hth by So is direct transcriptional repression is not clear. Ectopic So expression caused cell-autonomous repression of Ct and Hth, suggesting that the repression could be direct. Recently it was shown that So acts as a transcriptional repressor to repress ct transcription (Anderson, 2012). So may interact with a repressor and Groucho (Gro) is a likely candidate. So can bind to Gro and the So-Gro complex was postulated to repress Dac transcription in eye disc. The zebrafish So homologue Six3 interacts with Groucho and functions as a transcriptional repressor. The transcriptional co-repressor CtBP has been shown to functionally and physically interact with Ey, Dac and Dan. Whether the protein complex also involves So has not been determined. Overexpression of CtBP caused eye and antenna defect, but the phenotype was not affected by reducing so dose. Therefore, CtBP is probably not the co-repressor for So (Wang, 2012).

so3 clones caused non-autonomous induction of Ct in its surrounding wild-type cells. Similar non-autonomous induction of Dac has been reported in L3 disc. Elevated Delta was observed within the mutant clone and elevated activated N at the border of mutant clone, thus suggesting that the non-autonomous induction is due to N signaling to surrounding cells. Whether a similar mechanism operates in the L2 disc remains to be tested (Wang, 2012).

The finding that ey and toy do not repress Ct and Hth, in both gain-of-function and loss-of-function experiments, was initially perplexing. Clonal ey expression in the antenna disc did not repress Ct and Hth. In these clones, so-lacZ was induced, but not in all ey+ cells and at a level lower than the endogenous level in most cells in the eye disc. When ey was clonally induced at 29°C, Ct level was reduced. These results suggested that the ectopic ey and toy at 25°C induced so at a level not sufficient to repress Ct. The strength of Ey has been shown to be crucial for its ability to induce ectopic eye development. In the double knockdown of ey and toy in the eye disc, Ct and Hth were not induced. Judging from the eye disc phenotype and residual neuronal differentiation, the knockdown was not complete and may account for the failure to detect Ct and Hth derepression. Alternatively, additional factors, independent of ey and toy, may also repress Ct and Hth expression. This would be consistent with the weak effect of so3 clones in inducing Ct and Hth expression (Wang, 2012).

Hth expression is initially uniform in the eye-antenna disc but becomes restricted to the antenna disc in e-L2. In L3 eye disc, Hth expression is downregulated by Dpp and Hh, produced from the progressing MF and developing photoreceptors, respectively. However, Hth expression retracted from the posterior part of the eye disc in e-L2, even before the initiation of MF and photoreceptor differentiation. At e-L2, dpp and hh are expressed in the posterior region of the eye disc. It is possible that the early Hh and Dpp contributed to the repression of Hth from the eye disc, in addition to the repression by So (Wang, 2012).

The results showed that Ct and Hth represses ey transcription. The binding sites for both Hth and Ct in ey3.6 are required for its repression in the antenna disc, suggesting that both Hth and Ct bind to the ey3.6 enhancer directly. The ChIP assay results showed that both Hth and Ct can bind to the ChIP-1 fragment, which contains the binding site for Ct but not for Hth. This suggests that the Hth may bind through a Hth-Ct complex. However, as ectopic expression of either Hth or Ct is sufficient to repress ey transcription, the repression does not require the formation of the Hth-Ct complex (Wang, 2012).

In the RNAi experiments, knocking down Ct or Hth individually did not cause de-repression of the eye pathway genes. However, when the Ct- or Hth-binding site in ey3.6 was separately mutated, the repression of ey3.6 in the antenna disc was partially lost. One possible explanation for the discrepancy is that the RNAi knockdown was not complete. When the binding sites for both Ct and Hth were mutated, the de-repression of ey3.6 in the antenna disc was strongly enhanced. It is possible that both Ct and Hth contributed to the repression of ey transcription, and a threshold net amount of these repressors is required (Wang, 2012).

Hth physically interacts with Exd through the MH domain of Hth and the PBC-A domain of Exd to promote Exd nuclear localization. Hth generally acts as a transcriptional activator , but Hth and Exd can interact with En or UbxIa to repress transcription. Thus, Hth would need to interact with a repressor to repress ey. Ct can serve such a role. Ct can act as a transcriptional repressor by direct binding to a target gene. The human and mouse Ct homologues generally function as transcriptional repressor. However, as ectopic expression of Hth alone in the eye disc, in the absence of Ct, is sufficient to repress ey, Hth must be able to interact with an additional repressor (Wang, 2012).

This study found that Ct can also block the function of Ey when co-expressed with Ey. It is possible that the block resulted from the repression of toy transcription, which may reduce the strength of the feedback regulation of the retinal determination gene network (Wang, 2012).

Although Ct is expressed in L2 in the entire antenna disc, the phenotype caused by ct clones affected only restricted domains, perhaps owing to its later restricted expression. This study reports a novel function of Ct in antenna development. Ct and Hth function redundantly to repress the retinal determination pathway. Because of this functional redundancy, this Ct function was not revealed in ct clones (Wang, 2012).

hth or exd mutations caused antenna-to-leg transformation. Hth has a role in blocking eye development at the anterior margin of the eye disc, where Ct is not expressed. In the antenna disc, this function is masked because of the functional redundancy with Ct revealed in this study (Wang, 2012).

Even when both ct and hth were knocked down in their endogenous expression domain (hth>mi-hth+mi-ct), no significant transformation of the antenna to eye was seen in adult. One possible reason is that the hth>mi-hth+mi-ct caused lethality and the flies have to be raised at a lower temperature, thereby excluding a stronger phenotype. Another possibility is that the Dll expression in the antenna disc served to block eye development. Dll and hth are required in parallel for normal antenna development. Co-expression of Dll and hth can induce the formation of antenna structures in many ectopic sites. It may be the presence of Dll that blocked eye development and provided a leg identity to cause the distal antenna-to-leg transformation found in hth>mi-hth+mi-ct flies (Wang, 2012).

Targets of Activity

Regulation of the retinal determination gene dachshund in the embryonic head and developing eye: So targets dac

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).

Direct control of neurogenesis by selector factors in the fly eye: regulation of atonal by Ey and So

During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of 'eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. This study shows that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. These transcription factors interact in vitro and indirect evidence is provided that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation (Zhang, 2006).

This study found that regulatory elements controlling the early phase of ato expression in the eye lie within a 1.2 kb region located 3.1 kb downstream of the ato transcription unit. The early phase of ato transcription results from the integration of multiple regulatory inputs through separate cis-regulatory modules present within the 1.2 kb region (Zhang, 2006).

Cis-regulatory elements essential for gene activation map to the last 348 bp of the 1.2 kb region and include the So- and Ey-binding sites. Interestingly, the 348 bp region contains two relatively large (A1=99 bp and A2=140 bp) DNA sequences that are highly conserved from D. melanogaster to D. virilis. Based on this observation, constructs were generated containing only A1 or A2. However, neither region alone was sufficient to drive the stripe of reporter gene expression in the eye disc. Based on these results, it is conclude that the 348 bp region constitutes a 'core' or 'minimal' enhancer region for the transcriptional activation of ato in eye progenitor cells. Other factors undoubtedly bind to sequences within A1-A2 and regulate gene expression as neither A1 nor A2 alone are sufficient to drive expression in the eye disc. Genetic evidence suggests that signaling by the Bmp4-type factor Decapentaplegic (Dpp) also contributes to ato activation and two putative binding sites for Mad (a transcription factor shown to activate Dpp pathway targets) appear to be required for ato expression in all discs. However, a Mad consensus site present in the A2 box does not correspond to either of the two elements previously identified . Moreover, both the previously identified sites lie within the L fragment well upstream of the M'-M" interval containing the eye-disc enhancers. Future analyses of 3' enhancer-promoter interactions may resolve this issue (Zhang, 2006).

Separate cis-regulatory elements located within the conserved DNA regions IC1 and IC2 (IC1=88 bp and IC2=133 bp) control initial clusters formation. This feature of ato expression has been shown to require Notch (N) function. Sequence analysis of the IC1-IC2 region does identify a binding site for the effector of N signaling Suppressor of Hairless [Su(H)]. However, contradictory reports have been published on how Notch controls ato expression. Sun and colleagues found that transcription of ato is uniformly upregulated upon inactivation of Notch in Nts1 mutant discs. By contrast, early Ato protein expression is severely reduced in null Notch mutant clones . Since these experiments made use of different genetic reagents, it is difficult to interpret these results. Notch signaling may independently regulate ato expression at the mRNA and protein levels. Alternatively, the source of the discrepancy may lie in the use of different alleles, one hypomorphic (Nts1), and the other null (N54l9) (Zhang, 2006).

Lastly, activation of the 3'ato348gal reporter (core element) occurs prematurely as compared with endogenous ato. The 3'ato348gal mRNA is also found in cells lying just anterior to the proneural domain. Eye progenitors from this region are at a developmental stage referred to as pre-proneural and are characterized by the expression of the transcription factor Hairy (H) in addition to RD proteins. In the absence of Hairy and its partner Extra Macrochaetae, neurogenesis begins precociously within the eye disc. Thus, Hairy contributes to the downregulation of ato expression and prevents precocious neurogenesis. Activation of the reporters 3'ato348gal and 3'ato488gal (but not 3'ato1.2gal or 3'ato1.2-Δ298gal) in pre-proneural cells suggests that cis-elements mediating anterior repression lie within the 1.2 kb DNA fragment but outside the IC and A boxes. Although a search for canonical Hairy-binding sites does not identify potential regulatory elements, additional short stretches of evolutionarily conserved DNA are present and may contribute to this and/or other aspects of ato regulation (Zhang, 2006).

Over the last few years, Ey and So have been shown to play a crucial role in the deployment and maintenance of the RD network by directly regulating the transcription of several eye-specification genes [ey, so, eya, dachshund (dac) and optix]. However, little is known about downstream targets of the RD cascade. Although So also activates the post-MF expression of hedgehog and lozenge, this gene regulation is likely to reflect the late, differentiation-related functions of So. Thus, it is unclear how the RD factors induce eye formation and what aspects of the morphogenetic program they control directly (Zhang, 2006).

The results strongly suggest that the transcription factors Ey and So control activation of ato expression. This is the first example of a gene required during eye morphogenesis that is directly regulated by the RD network. The direct control of ato by Ey and So is a likely reason why ectopic eye induction by Eya+So or Dac depends on the activation of their upstream regulator ey. Other downstream targets may also be similarly controlled by multiple RD factors (Zhang, 2006).

The in vitro and in vivo evidence presented in this study also suggest that Ey and So may form a complex when bound to the adjacent cis-regulatory sites in the 3'ato core element. Together with the previously reported interactions of Eya-So and Eya-Dac, this finding raises the possibility that additional multimeric complexes involving several RD factors may also be involved in driving the transcriptional program for eye development. The observation that normal eye development is severely disrupted when one or another RD factor is over-expressed suggests that the RD proteins must be present at an appropriate level relative to one another. As all four proteins, Ey, Eya, So and Dac, have now been shown to interact in various combinations, the formation of such complexes and the recruitment of additional shared co-factors are likely to be sensitive to the relative concentration of RD factors present in eye progenitor cells (Zhang, 2006).

The model of gene regulation exemplified by the control of ato transcription provides a strong rationale for the feedback regulatory loops that link late and early RD gene expression. This regulation is likely to play a crucial role in ensuring the presence of appropriate levels of all four RD factors to optimize complex formation and co-regulation of downstream targets (Zhang, 2006).

Current models for the co-ordination of organ identity and neurogenesis in the eye place the Pax6 pathway either upstream of, or in parallel to, the control of neurogenesis. The findings presented in this paper favor the former model. Separate regions have been identified for the regulation of ato transcription in the eye versus other sensory organs (JO and CH). In addition, the presence of Ey- and So-binding sites that are required in vivo for reporter gene activation strongly suggests that endogenous ato expression is directly regulated by these factors. Thus, the RD network does not merely modify sensory organ development within the eye disc, but does, in fact, directly control it. In doing so, it also contributes to the co-ordination of selector and neurogenic inputs required to generate complex sensory structures such as the eye (Zhang, 2006).

Is this regulatory relationship between Ey-So and ato ancestrally derived? That is, was the direct link between ancestral Pax- and Ath-like genes already established in the protosensory organ that gave rise to today's ato-dependent sensory structures? The association of Pax-, Six- and Ath-type factors with sensory perception is not restricted to photic sensation but extends to mechanoreception in diverse organisms including mouse, jellyfish and mollusks. In the jellyfish P. carnea, which lacks eyes but responds to a variety of environmental stimuli including light, expression of a putatively ancestral-like PaxB gene, Six1/2, Six3/6 and atonal-like 1 is associated with neuronal precursors found in the medusa tentacles. Although the studies carried out in more basal metazoa consist mostly of analyses of gene expression and not function, this evidence does suggest that the association of Pax/Six/Ath-type factors and sensory organ development is ancient and may have been retained over more than 600 million years of evolutionary history (Zhang, 2006).

It is possible that the mechanisms of transcriptional regulation uncovered between Pax and Six genes and between Pax/Six and ato may have arisen early during evolution. Such regulatory interactions may have favored the continued association of Pax/Six/Ath as various modifications of their genetic cascades led to the development of more complex and diverse sensory organs. The investigation of ato/Ath gene regulation in other sensory organs and in basal metazoans is likely to clarify the evolutionary relationship among these pathways and the sensory modalities they control (Zhang, 2006).

Identification of transcriptional targets of the dual-function transcription factor/phosphatase eyes absent

Drosophila eye specification and development relies on a collection of transcription factors termed the retinal determination gene network (RDGN). Two members of this network, Eyes absent (EYA) and Sine oculis (SO), form a transcriptional complex in which EYA provides the transactivation function while SO provides the DNA binding activity. EYA also functions as a protein tyrosine phosphatase, raising the question of whether transcriptional output is dependent or independent of phosphatase activity. To explore this, microarrays together with binding site analysis, quantitative real-time PCR, chromatin immunoprecipitation, genetics and in vivo expression analysis were used to identify new EYA-SO targets. In parallel, the expression profiles of tissue expressing phosphatase mutant eya were examined, and it was found that reducing phosphatase activity did not globally impair transcriptional output. Among the targets identified by this analysis was the cell cycle regulatory gene, string (stg), suggesting that EYA and SO may influence cell proliferation through transcriptional regulation of stg. Future investigation into the regulation of stg and other EYA-SO targets identified in this study will help elucidate the transcriptional circuitries whereby output from the RDGN integrates with other signaling inputs to coordinate retinal development (Jemc, 2007).

Two general conclusions have resulted from this work: (1) the similarity in expression profiles between tissue overexpressing wild type and phosphatase-dead eya transgenes and analysis of gene expression by quantitative PCR suggest that EYA's phosphatase is not generally required for EYA transcriptional activity, although it may be required for maximal transactivation of some target genes, and (2) the short sequence (T/C/G)GA(A/T/G)A(T/C) appears to be the only recognizable motif shared among all SO binding sites. As exemplified by in vivo validation of EYA-SO-mediated regulation of the cell cycle regulator stg, further analysis of the target genes identified in this study will likely shed new light into the mechanisms underlying EYA-SO function during development (Jemc, 2007).

The main goal of this study was to identify new targets of EYA transcriptional activity. Although adult head tissue was used for overexpression experiments, an 86% success rate in confirming changes in expression of potential targets in developing Drosophila eye-antennal imaginal discs overexpressing eya supports the ability of this system to identify similar data sets in different developmental stages. Out of the ten genes upregulated by eya overexpression in both adult head tissue and eye-antennal imaginal discs, five demonstrated enrichment of endogenous SO at one or more predicted binding sites. These predicted binding regions were conserved across a minimum of two and up to nine Drosophila species, emphasizing their likely biological relevance. Two binding sites that did not demonstrate SO enrichment were not conserved across other Drosophila species, while binding sites in the remaining three genes were conserved across multiple species and could be EYA-SO targets in other tissues (Jemc, 2007).

The core sequence shared by all of these targets is (T/C/G)GA(A/T/G)A(T/C), a pared down version of the previously proposed GTAAN(T/C)NGANA(T/C)(C/G) SO binding sequence. In Drosophila EYA-SO targets, the sequence flanking the core (T/C/G)GA(A/T/G)A(T/C) has been shown to be important only in the case of the target so, and is absent in one of the two SO binding sites identified in the lz locus and the binding sites in stg were confirmed by gel shifts. While specific flanking sequences may further stabilize SO-DNA interactions, characterization of such a flanking sequence consensus awaits further analysis. Confirmation of additional targets predicted by microarray and binding site analysis should provide for further characterization of the SO binding sequence. Out of the remaining 31 potential targets, all except one have binding sites conserved across multiple Drosophila species, suggesting that additional EYA-SO targets will be confirmed within this data set (Jemc, 2007).

While none of the previously identified EYA-SO targets were included in the final list, two targets, so and lz were upregulated upon eya overexpression, although less than the two-fold cutoff. The expression of the previously identified targets hh, ato and ey was either absent or changes were not statistically significant. One explanation for this observation is that other signaling pathways required for the expression of these genes may not be activated, or, conversely, inhibitory signaling pathways could be activated in adult head tissue (Jemc, 2007).

In addition to examining expression levels of previously identified EYA-SO targets, the list of upregulated EYA-SO target candidates was compared to genes that were upregulated by ey overexpression in microarray analyses. Because ey both induces eya and so expression and is itself transcriptionally regulated by EYA-SO, one would expect to see a number of genes similarly regulated by overexpression of either ey or eya; however, as detailed below, pairwise comparisons between the current data set to lists of candidate ey targets derived from two independent array studies, reveals a surprisingly limited overlap. One study identified 371 genes with at least 1.5-fold upregulation across two array experiments, only 55 of which were similarly upregulated in both arrays. Comparison of this data set to a second more recent report of 300 candidate genes upregulated in response to ey overexpression revealed only 24 common targets. Comparison to the current list of potential eya-so targets yielded 10 shared with the first data set and 3 common to the second results. Encouragingly, despite this limited overlap, stg, a gene shown in this study to be transcriptionally regulated by eya and so, was one of the two targets consistently upregulated in all three studies (Jemc, 2007).

As additional targets are confirmed, it is important to note that EYA may also associate with transcription cofactors other than SO to regulate gene expression. Although EYA can associate with DAC, and X-ray crystallographic analysis suggests DAC can bind DNA, targets of an EYA-DAC complex or a consensus DAC binding site have not been identified. In addition, EYA also contains an engrailed homology 1 (eh1) domain, suggesting it may be able to bind to the transcriptional repressor Groucho (GRO). However, as current in vivo data only supports a role for EYA as a transactivator complexed to SO, identification of additional EYA cofactors in vivo will be necessary to explore the potential of SO-independent EYA transcriptional functions further (Jemc, 2007).

Many of the genes identified as direct EYA-SO transcriptional targets are largely uncharacterized 'CGs' whose expression patterns in the eye will have to be studied in detail to gain further insight to EYA-SO-mediated regulation, but a few have predicted or well-studied functions that may provide insight into how EYA-SO functions during normal development and how misregulation can result in disease. Most notable on this list is stg. Given that overexpression of eya and so results in overproliferation, while their loss leads to tissue reduction, EYA-SO control of stg expression provides a mechanism for how EYA-SO regulation of the cell cycle may in turn affect cell proliferation. An interesting question for future investigation is how the relatively broad expression of EYO and SO throughout the developing retina activates stg expression only in a relatively narrow stripe of cells just anterior to the morphogenetic furrow. Given the apparent complexity of stg cis-regulatory elements, a likely explanation is that EYA-SO act combinatorially with transcriptional effectors of other signaling pathways to effect this developmental precision (Jemc, 2007).

Consistent with eya and so overexpression leading to increased tissue overgrowth in Drosophila, elevated levels of Eya and Six family members have been observed in a variety of cancers. Studies of the transcriptional targets of mammalian Eya and SO/Six proteins have identified the cell cycle regulatory genes, cyclin D1 and cyclin A1, the proto-oncogene c-Myc and ezrin, a regulator of the cytoskeleton and contributor to metastasis, suggesting intermediates through which Eya and Six family genes regulate proliferation and contribute to cancer. Identification of stg as a transcriptional target of EYA and SO in Drosophila provides not only the first direct cell cycle target in Drosophila, but also suggests another target through which EYA and SO might regulate proliferation in other organisms (Jemc, 2007).

Before parallels can be drawn to how EYA-SO targets important for Drosophila retinal development might be relevant to development and disease in other organisms, it will be necessary to examine the conservation of the transcriptional regulatory circuits. However, given the predicted functions of the gene products encoded by candidate EYA-SO targets, together with knowledge of Eya-Six function in mammalian systems, it is tempting to speculate. For example, CG12030, the Drosophila homolog of the human Gale, encodes a sugar epimerase required for galactose metabolism. As metabolic abnormalities have been demonstrated to play a part in cataract formation, and mutations in eya have been observed in patients with congenital cataracts, the identification of CG12030 as an EYA-SO target suggests intermediates through with eya might function to maintain homeostasis in the eye. Mal, which encodes a molybdenum cofactor sulfurase important for ommochrome biosynthesis, is expressed in Drosophila pigment cells in the eye and would seem a logical target of the RDGN. Mutations of the human homolog of mal, HMCS, can result in renal failure and myositis, both intriguing phenotypes given the importance of Eya-Six in vertebrate kidney and muscle development. CG15879 encodes the Drosophila homolog of human SERHL2, a member of a serine hydrolase-like family predicted to regulate muscle growth, a developmental context in which Eya and Six family genes function in Drosophila and vertebrates. Lastly, CG8449 has a predicted RabGAP/TBC domain. While RabGAPs function in a variety of developmental contexts, RabGAP-like proteins have been predicted to function in phototransduction and synaptic transmission in Drosophila and mutations in RabGAP genes have been isolated in cases of Warburg Micro syndrome, a severe autosomal recessive disorder characterized by abnormalities in the eye, as well as the central nervous system and genitals, all contexts in which Drosophila eya and so are expressed. Identification of additional EYA-SO targets and the examination of the conservation of EYA-SO transcriptional regulation across homologous genes in different species will be necessary to determine how EYA and contribute to development and disease (Jemc, 2007).

Given the importance of achieving appropriate levels of gene expression during the course of development, it is not surprising that multiple signaling pathways converge to regulate common target genes at the level of transcription. For example, hh and lz are coordinately regulated by receptor tyrosine kinase (RTK) downstream effectors of the ETS family in conjunction with EYA and SO. This study has identified stg as an EYA-SO target and suggests stg transcription is also regulated Notch and Wingless (Wg) signaling, and by RTK signaling. Thus, these results suggest a mechanism by which members of the RDGN are integrated with Notch and Wg signaling to coordinate cell proliferation. Identification of additional EYA-SO targets is likely to reveal new nodes for integration of the RDGN with other signaling pathways, explaining how signaling pathways cooperate to yield specific developmental outcomes (Jemc, 2007).

Direct control of the proneural gene atonal by retinal determination factors during Drosophila eye development

The determination of neuronal identity in Drosophila cells depends on the accurate expression of proneural genes. The proneural gene atonal (ato) encodes a basic-HLH protein required for photoreceptor and chordotonal organ formation. The initial expression of ato in imaginal discs is regulated by sequences that lie 3' to its open reading frame. This report shows that the initial ato transcription in different imaginal discs is regulated by distinct 3' cis-regulatory sequences. The eye-specific ato 3' cis-regulatory sequence consists of two distinct elements termed 2.8PB and 3.6BP that regulate ato transcription during different stages of eye development. The 2.8PB enhancer contains a highly conserved consensus binding site for the retinal determination (RD) factor Sine oculis (So). Mutation of this So binding site abolishes 2.8PB enhancer activity. Furthermore the RD factors So and Eyes absent (Eya) are required for 2.8PB enhancer activity and can induce ectopic 2.8PB reporter expression. In contrast, ectopic Dpp signaling is not sufficient to induce ato 3' enhancer activation but can induce increased levels of RD factor Dachshund (Dac) and synergize with So and Eya to increase ato 3' enhancer activity. These results demonstrate a direct mechanism by which the RD factors regulate ato expression and suggest an important role of Dpp in the activation of ato 3' enhancer is to regulate the levels of RD factors (Tanaka-Matakatsu, 2008).

In addition to RD factors, Dpp signaling is also known to be involved in eye development although little is known about its role in the activation of the ato 3′ enhancer. This study found that induction of the ato 3′ enhancer by ectopic expression of So and Eya under the 30A-GAL4 driver was limited mainly to specific regions near the A/P compartment boundary where endogenous Dpp is expressed. In addition, co-expression of Dpp with So and Eya led to expansion of ectopic ato 3′ reporter expression, indicating that Dpp signaling can synergize with So and Eya to activate the 2.8PB enhancer. As the 2.8PB enhancer does not contain Mad binding sites, it is unlikely that Dpp signaling regulates 2.8PB expression directly through binding of Mad protein to 2.8PB. It is hypothesized that some of the downstream targets of Dpp signaling may mediate the ability of Dpp signaling to synergize with So and Eya in the activation of the ato 3′ eye enhancer. Interestingly, Dac, a RD factor regulated by Dpp signaling, can also synergize with So and Eya in activating the ato 3′ eye enhancer, raising the possibility that induction of Dac contributes to the ability of dpp to synergize with so and eya in the activation of ato 3′ enhancer. The level of Dac in the posterior of the wing disc is significantly lower than that in the anterior in the absence of Dpp co-expression, while similar levels of Dac in the anterior and the posterior are observed when Dpp is co-expressed. Therefore the difference in the subset of cells induced to activate the ato 3′ enhancer by dpp + so + eya and by dac7c4 + so + eya expression could be in part due to differences in the level of Dac induced by Dpp expression and that reached with the 30A-GAL4 driver. Alternatively, it is possible that Dpp signaling has additional targets that contribute to its synergistic induction of the ato 3′ enhancer with So and Eya (Tanaka-Matakatsu, 2008).

During Drosophila sensory organ formation, transcriptional regulation of the proneural gene ato plays a key role to determine the position of proneural clusters. Tissue-specific expression of ato is governed by the flanking cis-regulatory regions immediately upstream (5′) and downstream (3′) of the ato transcription unit. ato 5′ transcription largely depends on the Ato-dependent autoregulatory mechanism, while the ato 3′ cis-regulatory region appears to encode tissue- and temporal-specific information. This analysis of the ato 3′ cis-regulatory region revealed a modular organization of tissue-specific enhancers, each of which determine the initial ato expression in sensory organ precursors of a specific tissue type for the formation of ch organs or photoreceptors. For example, the 1.7 kb BamHI–StuI fragment immediately downstream of the ato transcription unit controls ato expression specifically in the leg discs while the 1.9 kb StuI–PstI fragment located 1.7 kb downstream of the ato transcription unit regulates ato expression specifically in the antennal ch organ precursors. Similarly, the eye enhancer lies within the BglII–PstI–EcoRI fragment located 2.8 kb downstream of the ato transcription unit. Finally the 1.5 kb EcoRI–BamHI fragment located 4.8 kb downstream of the ato transcription unit regulates ato expression during embryonic development (Tanaka-Matakatsu, 2008).

Taken together, these results demonstrate that the modular organization of the ato 3′ cis-regulatory region determines the spatial control of ato expression in the ch organs and photoreceptors in different imaginal discs. A surgical experiment of eye disc fragments has revealed that cells immediately anterior to the MF have already acquired the potential to differentiate into retina. Cells ahead of the MF express RD genes and anti-proneural genes to precisely control retinal cell fate determination and proneural cell differentiation. This region is referred to as the pre-proneural (PPN) domain, based on competence for retinal differentiation. The observation that the 2.8PB but not the 6.4BB enhancer os activated precociously in the PPN region suggests the presence of repressor elements residing within the 3.6BP fragment that contribute to the timing of atonal activation during MF progression. Interestingly, gain of function experiments in the wing disc did not reflect significant differences between 2.8PB and 6.4BB. Both enhancers conferred reporter expression only in groups of cells near the A/P compartment boundary in response to So and Eya and co-expression of dpp with so and eya led to an expansion of GFP expression mostly in the posterior domain. It is possible that some positive and negative factors required for the proper regulation of the ato 3′ enhancer in eye discs were not present in the wing disc. Previous studies have identified a number of genes sufficient to induce retinal tissue development or precocious photoreceptor differentiation, and these genes are potential candidates that contribute to the precise expression of ato. For example, ectopic expression of eyegone (eyg) or Optix (Optx) induces retinal tissue development while induction of mutant clones for either extradenticle (exd) or homothorax (hth) lead to ectopic eye formation in the ventral head region. Additionally, ectopic activation of the Hh signaling pathway or removal of hairy (h)/extramacrochaetae (emc) is sufficient to induce precocious furrow advancement and photoreceptor differentiation. Furthermore, removal of the Notch effector Su(H) causes slight advancement of neural differentiation. This search of conserved non-coding DNA sequences did not find predicted Ci binding sites in the ato 3′ cis-regulatory region. In contrast, a highly conserved transcription factor binding site for Su(H) is observed in the ato 3′ cis-regulatory region. Further analysis of ato 3′ eye enhancer should help to define the mechanisms that contribute to the precise control of its expression (Tanaka-Matakatsu, 2008).

Onset of atonal expression in Drosophila retinal progenitors involves redundant and synergistic contributions of Ey/Pax6 and So bindings sites within two distant enhancers

Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. This study shows that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). The study shows how these sites interact to induce ato expression in distinct regions of the eye field and confirms them to be occupied by endogenous Ey and So proteins in vivo. This study suggests that Ey and So operate differently through the same 3' cis-regulatory sites in distinct populations of retinal progenitors (Zhou, 2013).

Protein Interactions

So and Eyes absent are able to physically interact through their evolutionarily conserved domains. The sequences responsible for interaction are localized to the N-terminal domain of Eya, while the So interaction domain is localized to the Six domain, a conserved sequence shared with vertebrate So homologs. Because of the multiple effects of the So/Eya interaction in MF induction, cell proliferation and neural induction, it is proposed that a So/Eya complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity (Pignoni. 1997).

The eyes absent gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other proteins of eye development, Sine oculis and Dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, eya alleles have been sequenced. Loss of the entire Eya Domain is null for eya activity, but alleles with truncations within the Eya Domain display partial function. The molecular genetic analysis was extended to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminish the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it (Bui, 2000).

This analysis of the mutations in the Eya Domain was extended to the situation in vivo by generating transgenics expressing the selective point mutants that disrupt interactions with So and Dac in the yeast two-hybrid system. Although this has failed to provide evidence in support of a special functional relevance of the Dac interaction (both mutant forms appeared to interact similarly in ectopic eye formation upon coexpression with Dac) evidence has been found supporting the importance of the So interaction. These data indicate that the EyaE11 mutant form shows a diminished ability to synergize with So upon coexpression. This supports the hypothesis that the eyaE11 mutation within the Eya Domain disrupts interactions in vivo with so (and/or possibly with other Six homologs) that are critical for the function in eye formation. The EyaE7 mutant form, which shows a disrupted Dac interaction, still supports ectopic eye formation, although at decreased penetrance compared to normal Eya. dac null mutations frequently show some degree of eye development, suggesting that dac may be partially redundant in eye formation. Therefore, even if interaction with Dac in vivo were disrupted by the eyaE7 mutation, eye formation might still occur due to compensation by such mechanisms. Nevertheless, this eya allele also shows a dominant reduced-eye phenotype when coexpressed with so -- this is a new property not observed with the wild-type Eya protein. The eyaE7 mutation may generate a protein with some dominant-negative property in eye formation. The data that So and Dac may interact, in part, differentially within the conserved domain of Eya supports the idea that the three proteins have the potential to interact in a single complex in vivo. Such a hypothesis, however, is complicated by other data indicating that the molecular activity of Eya-So coexpression in eye formation is at least in part distinct from that of Dac or Eya-Dac coexpression: whereas Dac, and Eya coexpression with Dac, activate an eya enhancer, Eya alone or Eya with So fails to activate enhancer activity, despite ectopic eye formation (Bui, 2000).

Fly SIX-type homeodomain proteins Sine oculis and Optix partner with different cofactors during eye development

Two members from the Six class of homeobox transcription factors, Sine oculis (SO) and Optix, function during development of the fly visual system. Differences in gain-of-function phenotypes and gene expression suggest that these related factors play distinct roles in the formation of the fly eye. However, the molecular nature of their functional differences remains unclear. This study reports the identification of two novel factors that participate in specific partnerships with Sine oculis and Optix during photoreceptor neurons formation and in eye progenitor cells. This work shows that different cofactors likely mediate unique functions of Sine oculis and Optix during the development of the fly eye and that the repeated requirement for SO function at multiple stages of eye development reflects the activity of different SO-cofactor complexes (Kenyon, 2005).

The fly SIX-HD transcription factors SO and Optix function during eye development and are expressed in different but overlapping patterns within the eye epithelium. Targeted expression of SO or Optix results in distinct phenotypes. Optix is able to induce eye development when ectopically expressed in the antenna and blocks eye morphogenesis when overexpressed within the eye epithelium. On the contrary, SO does not induce ectopic eye formation nor does it block neuronal differentiation within eye tissue. These observations strongly suggest that SO and Optix fulfill different roles during eye development. The identification of multiple unique partners for these Six factors clearly supports a model in which differences in cofactor recruitment contribute significantly to their specific function. This paper describes the identification of two novel potential partners of SO and Optix: one that is SO-specific by virtue of its expression pattern, and another likely to be Optix-specific by virtue of its protein binding profile (Kenyon, 2005).

The Sbp gene (Flybase ID FBgn0033654) encodes a novel factor of unknown function. It does, however, contain three highly conserved motifs and a proline-rich region. The high level of similarity from fly to vertebrates suggests that these sequences represent novel protein motifs or domains with specific functions. Since they do not appear related to previously characterized domains, little can be concluded about their potential function. However, Box 1 is not required for binding to the SD of SO, because the clone isolated through the yeast two-hybrid screen does not include this sequence. Its interaction with SO suggests that Sbp may function as a transcriptional regulator. The presence of a proline-rich region supports this hypothesis because proline-rich domains have been implicated in both transcriptional activation and repression. In a manner similar to Gro and unlike Eya, Sbp can interact in yeast with both SO and Optix. However, Sbp is specifically coexpressed with SO and not Optix within the eye epithelium. Hence, its interaction in vivo is restricted to SO by virtue of its expression posterior to the MF. This hypothesis is consistent with the observation that ectopic expression of Sbp in progenitor cells, where Optix is also expressed, interferes with normal eye development. However, alternative explanations are also possible. The effect of Sbp misexpression may be due to its abnormal association with SO itself, or other nuclear factors, in progenitor cells. Similarly, the disruption of eye development due to the misexpression of Optix in the developing neuronal field may result from abnormal interactions with other yet unidentified factors. Nonetheless, restricted expression of Sbp posterior to the MF and Optix anterior to the MF is obviously critical for normal eye development and effectively excludes binding between Optix and Sbp (Kenyon, 2005).

Sbp expression within the developing neuronal array, and not in progenitor cells, indicates that this gene is not required at an early stage but functions at the time of neuronal differentiation. On the contrary, SO function is required at multiple stages of eye development, including during specification of eye progenitor cells, at the time of patterning (in the MF) and during neuronal development. The Eya cofactor has also been implicated in these steps, and both proteins are continuously expressed in eye tissue through these developmental stages. These studies do not provide clues regarding the function of SO at each developmental stage. Based on these data, SO could function simply by driving expression of a few genes required at all times to maintain eye identity. The identification of an SO partner specifically expressed in the differentiating epithelium contradicts this hypothesis and shows that there are significant differences in at least some of the SO-based complexes that function at distinct developmental stages. Thus, posterior to the MF, Sbp may associate with the SO-Eya complex and modify its activity turning it from an activator into a repressor. Evidence of complex interactions between the mouse Six1, Dac, and Eya proteins has been described. Alternatively, Sbp may form a distinct SO-Sbp complex. In either case, however, SO function at various times during eye development would be modulated by its interaction with specific partners (Kenyon, 2005).

The Obp gene (Flybase ID FBgn0050443) encodes a protein characterized by nine putative zinc-finger domains of the C2H2 type. Although most members of this family are thought to regulate transcription through DNA binding, recent reports have also implicated C2H2-type zinc-fingers in protein-protein interactions. Because zinc-fingers 4 through 9 as well as the C-terminal sequence of Obp are present in the fragment isolated in the screen, it cannot be excluded that one or more of these zinc-finger domains mediate the interaction with the Optix transcription factor. Obp is coexpressed with both SO and Optix in eye progenitor cells. However, it behaves as an Optix-specific interactor in yeast two-hybrid tests and does not impair eye development when ectopically expressed in the differentiating neuronal field (where only SO is present). Hence, this factor is likely to be a specific partner of Optix in vivo (Kenyon, 2005).

This work has not identified clear homologues of this factor. This finding is not entirely unexpected, because zinc-finger domains display only low-level conservation at positions other than the C and H residues that bind zinc. In the related proteins from D. pseudoobscura and A. mellifera, conservation is restricted to the region encoding zinc-fingers 4 through 8. Hence, additional criteria (such as expression in eye progenitor cells, interaction with Optix, and/or mutant rescue) are required to assess whether they are indeed homologues. Obp is reported in Flybase to encode a product putatively involved in cell proliferation. In fact, the fourth and sizth zinc-fingers show similarity to zinc-fingers present in the Sfp1 transcription factor. In yeast, Sfp1 functions in cellular growth and proliferation by regulating ribosomal proteins expression. Obp expression, specifically anterior to the MF where undifferentiated progenitors proliferate and then arrest just before the start of eye morphogenesis, is consistent with a potential role in regulating cell proliferation. However, the alignment between Obp and Sfp1 is limited to less than 20% of what is currently defined as the Sfp1 domain. Given the limited conservation and the lack of functional data, a role for Obp in proliferation control is speculative at this time (Kenyon, 2005).

sine oculis: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1998 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.