clift/eyes absent


cis-Regulatory Sequences and Functions

Drosophila eyes absent is required for normal cone and pigment cell development

In Drosophila, development of the compound eye is orchestrated by a network of highly conserved transcriptional regulators known as the retinal determination (RD) network. The retinal determination gene eyes absent (eya) is expressed in most cells within the developing eye field, from undifferentiated retinal progenitors to photoreceptor cells whose differentiation begins at the morphogenetic furrow (MF). Loss of eya expression leads to an early block in retinal development, making it impossible to study the role of eya expression during later steps of retinal differentiation. Two new regulatory regions have been developed that control eya expression during retinal development. These two enhancers are necessary to maintain eya expression anterior to the MF (eya-IAM) and in photoreceptors (eya-PSE), respectively. Deleting these enhancers affects developmental events anterior to the MF as well as retinal differentiation posterior to the MF. In line with previous results, reducing eya expression anterior to the MF was found to affect several early steps during early retinal differentiation, including cell cycle arrest and expression of the proneural gene ato. Consistent with previous observations that suggest a role for eya in cell proliferation during early development, deletion of eya-IAM was found to lead to a marked reduction in the size of the adult retinal field. On the other hand, deletion of eya-PSE leads to defects in cone and pigment cell development. In addition it was found that eya expression is necessary to activate expression of the cone cell marker Cut and to regulate levels of the Hedgehog pathway effector Ci. In summary, this study uncovers novel aspects of eya-mediated regulation of eye development. The genetic tools generated in this study will allow for a detailed study of how the RD network regulates key steps in eye formation (Karandikar, 2014, PubMed ID: 25057928).

Targets of activity

Ectopic expression of sina oculis has little or no effect on antennal, wing, or leg disc development, while ectopic eya expression often causes mild growth alterations resulting in extra folds in the epithelium and, rarely, formation of small ectopic ommatidial arrays in the antennal disc. Coexpression of so and eya leads to a dramatic increase in the development of ectopic eye tissue in antennal discs. These ommatidial arrays lead to adult eye structures. Ectopic so/eya induce eyeless expression in the antennal disc. When expressed in eyeless mutant discs, ectopic so/eya produces growth alterations, but ectopic eyes are not observed (Pignoni, 1997).

The eyeless, dachshund, and eyes absent genes encode conserved, nuclear proteins that are essential for eye development in Drosophila. Misexpression of eyeless or dachshund is also sufficient to induce the formation of ectopic compound eyes. Like ey and dac, targeted expression of eya alone is sufficient to induce ectopic eye formation. However, in contrast to ey, the penetrance of the ectopic eye phenotype induced by either dac or eya alone is incomplete and, when induced, such eyes are small. When dac expression was strongly induced in all imaginal discs, ectopic eye development was observed only on the anterior surface of the fly head ventral to the antenna, in just 56% (61/109) of animals examined. In contrast to the low penetrance of ectopic eye formation induced by dac or eya expressed alone, coexpression of dac and eya induces substantial ectopic eyes on the head, legs, wings, and dorsal thorax of 100% of animals examined. On the head, the cuticle between the normal eye field and antennae is transformed into retinal cells such that the normal retinal field is expanded. Large patches of pigment are induced on the dorsal surface of the femur and tibia of all legs, which are severely truncated. Ectopic eya alone can induce small patches of glass expression in the pouch area of the wing disc with 25% penetrance. In no case has ectopic Glass staining been observed in leg discs with either dac or eya alone. However, when dac and eya are coexpressed, ectopic Glass staining is induced with 100% penetrance along the ventral margin of the eye-antennal disc, the dorsal half of the leg disc along the anterior-posterior compartment (A/P) boundary, and along the A/P boundary of the dorsal wing disc. In each case, the sites of ectopic glass expression in discs correspond to the positions of ectopic retinal development observed in adults. Taken together, these data demonstrate that dac and eya show strong genetic synergy to induce ectopic retinal development in Drosophila (Chen, 1997).

Ectopic Elav-positive cells are induced in the antennal, leg, and wing discs, in response to dac and eya coexpression, suggesting ectopic neural differentiation. These ectopic neurons must be photoreceptor cells, since the visual system-specific Glass protein is also induced in the same pattern. Ectopic eyes observed in adults corresponding to these positions contain all of the normal cell types associated with the wild-type eye, including pigment cells, lens-secreting cone cells, and interommatidial bristles. The ectopic neurons induced by dac and eya misexpression send out axonal projections. The axons of ectopic photoreceptors in the eye-antennal disc form a bundle that extends posteriorly into the eye imaginal disc. These axons appear to fuse with the axon tracts sent out by photoreceptors of the normal retinal field that exit through the optic stalk to synapse in the brain. It is likely, therefore, that the fly can perceive light through ectopic photoreceptors formed in the eye-antennal disc as a result of dac and eya coexpression. Coexpression induces ectopic dpp expression in the eye-antennal disc adjacent to the field of ectopic photoreceptors. In the leg disc, dpp expression is split and forms a ring around the ectopic photoreceptors, again suggesting that an ectopic MF is initiated and propagates (Chen, 1997).

While eya expression in the eye disc does not depend on dac function, dac expression is greatly reduced in an eya2 mutant background, demonstrating that dac expression requires eya activity. Similarly, eyeless (ey) induction of ectopic dac expression is greatly reduced in an eya2 mutant background. These results suggest that dac may function downstream of eya. Consistent with this interpretation, eya is unable to induce ectopic eye formation in a dac mutant background. eyeless misexpression is sufficient to induce eya, suggesting that eya may be required for ey function. Indeed, ectopic retinal development driven by targeted ey expression fails to occur in an eya2 mutant background. Induction of eya expression by ey does not depend on dac activity, consistent with the idea that eya functions downstream of ey but upstream of dac. However, these genes do not act in a simple, linear pathway; targeted expression of dac and eya strongly induce the expression of one another, and eya is required for ectopic eye induction by dac. Misexpression of dac or eya is also sufficient to induce ectopic ey expression in the antennal disc. These results suggest that multiple positive-feedback loops exist among these genes during normal eye development and raises the possibility that ey may be required for ectopic retinal induction by eya and dac. Indeed, ectopic eye formation driven by coexpression of dac and eya is completely blocked in an eyeless2 mutant background, indicating that induction of ey is essential. It is proposed that a conserved regulatory network, rather than a linear hierarchy, controls retinal specification and involves multiple protein complexes that function during distinct steps of eye development (Chen, 1997).

Signaling by the secreted Hedgehog, Decapentaplegic and Wingless proteins organizes the pattern of photoreceptor differentiation within the Drosophila eye imaginal disc; hedgehog and decapentaplegic are required for differentiation to initiate at the posterior margin and progress across the disc, while wingless prevents it from initiating at the lateral margins. Wingless and Decapentapegic often have opposing functions in differentiation. This is true in the eye imaginal disc, where Dpp effects are positive, promoting initiation of the morphogenetic furrow and also promoting photoreceptor differentiation. Wg effects are negative, opposing the effects of Dpp, inhibiting furrow initiation and likewise inhibiting differentiation. An analysis of these interactions has shown that initiation requires both the presence of decapentaplegic and the absence of wingless, which inhibits photoreceptor differentiation downstream of the reception of the decapentaplegic signal. The eyes absent and eyegone genes encode members of a group of nuclear proteins required to specify the fate of the eye imaginal disc. Both eyes absent and eyegone are required for normal activation of decapentaplegic expression at the posterior and lateral margins of the disc and also repression of wingless expression in presumptive retinal tissue. The requirement for eyegone can be alleviated by inhibition of the wingless signaling pathway, suggesting that eyegone promotes eye development primarily by repressing wingless. These results provide a link between the early specification and later differentiation of the eye disc (Hazelett, 1998).

As a direct test of the requirement for dpp in furrow initiation and of the inhibitory role of wg, clones of cells were generated of doubly mutant for Mothers against dpp (required to transduce the dpp signal) and wg were examined. Cells in these clones are unable to respond to dpp, but are also unable to produce wg. When such clones of cells occur at the posterior margin of the eye disc, they autonomously fail to initiate photoreceptor development. Clones of cells singly mutant for Mad also fail to differentiate as photoreceptors, but often have an additional non-autonomous inhibitory effect on photoreceptor differentiation by surrounding cells. This inhibitory effect is likely to be mediated by wg. Thus dpp signaling is required not only to repress wg expression, but also independently for morphogenetic furrow initiation (Hazelett, 1998).

wg is shown to inhibit photoreceptor formation downstream of the dpp receptors. wg is known to be required to prevent ectopic morphogenetic furrow initiation from the lateral margins of the eye disc. However, the mechanism by which wg inhibits photoreceptor differentiation is not well understood. It has been suggested that wg acts by preventing dpp expression, since dpp expression is lost in clones of cells lacking the kinase encoded by shaggy/zeste-white 3 (sgg), which normally functions to inhibit the wg pathway. However, a low level of ectopic wg can inhibit photoreceptor differentiation without reducing dpp expression. Since dpp positively autoregulates its own expression, inhibition of dpp function may result in a loss of dpp expression. If wg acted by inhibiting dpp expression, it should be possible to overcome its effects by expressing dpp from a heterologous promoter. However, co-expression of dpp and wg does not allow initiation of photoreceptor development at the posterior margin. Ectopic expression of dpp in the eye disc has been shown to specifically induce initiation of photoreceptor differentiation from the anterior margin of the disc in a non-autonomous fashion. Surprisingly, this ectopic differentiation is not inhibited by wg signaling. Co-expression of dpp and wg throughout the disc results in initiation from the anterior margin at a much higher frequency than from the posterior margin. Thus initiation from the anterior margin must be able to overcome the inhibition normally caused by wg. wg inhibitory function is shown to act downstream of or in parallel to the action of Thickveins, a receptor for Dpp (Hazelett, 1998).

Rather than affecting the Dpp pathway directly, Wg might block photoreceptor differentiation at a stage subsequent to Dpp signaling. Formation of all photoreceptors is known to depend on the EGF receptor and its downstream component Ras. Wg has recently been shown to antagonize EGF receptor signaling during the specification of the cuticle pattern in the embryo. To determine whether wg also acts on this pathway in the eye, a test was performed to determine if a secreted and active form of the ligand Spitz (s-Spi) or a constitutively active form of Ras could bypass the block caused by wg. In discs expressing both Wg and activated Ras ubiquitously, extensive photoreceptor differentiation and growth are observed, as in discs expressing activated ras alone. Thus Wg must act upstream, prior to Ras activation, to block differentiation. Expression of s-Spi also rescues photoreceptor differentiation in discs expressing Wg ectopically. The inhibition of photoreceptor differentiation is mediated by the conventional Wg signal transduction pathway, because a constitutively active form of Armadillo, a mediator of the Wg signal, is shown to block photoreceptor differentiation (Hazelett, 1998).

Since the phenotype caused by ectopic wg is rescued by expressing activated forms of Spi or Ras it is possible that Wg interferes with Egf receptor signaling upstream of (prior to the activation of) Ras. Recently, it has been shown that in the embryonic segments Wg and secreted Spi emanate from distinct sources and promote opposing cell fates. This led to the proposal that Wg antagonizes signaling by Spi through the EGF receptor and the Ras/MAPK cascade. Since EGF receptor signaling is required for the formation of all photoreceptors, it is a possible target for Wg inhibition in the eye disc. However, it does not appear that the effects of ectopic Wg can be completely explained by the antagonism of Spi signaling, since mutations in spi allow the specification of R8 and the progression of the furrow, while the presence of ectopic Wg does not. It is possible that another ligand, such as Vein, normally activates the EGF receptor in R8 and that this ligand is also antagonized by Wg. Another possibility is that Ras activation in R8 is mediated by another tyrosine kinase receptor; one of the identified FGF receptors is expressed in the morphogenetic furrow. The lower effectiveness of rescue by s-Spi than by activated Ras also suggests that Wg has effects both upstream of Spi expression or processing, and downstream of these events. Some factors known to be required between Spi and Ras that could be targets of Wg inhibition are Daughter of sevenless, Downstream of receptor kinases and Son of sevenless. Alternatively, Wg could act by stimulating the expression or function of Argos, a secreted antagonist of Spi (Hazelett, 1998).

Genes implicated in early events of eye development, eyes absent and eyegone, are involved in the regulation of dpp and wg expression. The expression of dpp and wg were examined in eya mutant eye discs. Expression is greatly reduced in early third instar eya mutant discs, prior to the initiation of the morphogenetic furrow, and was completely lost in eya mutant clones, suggesting that eya is required for dpp transcription. Although the initiation of wg expression in early eya mutant eye discs appeared to be normal, ectopic Wg protein was observed in eya mutant clones in late third instar discs. Another gene required for eye formation that has not been placed within the hierarchy of eye development is eyegone (eyg); in its absence, no photoreceptors differentiate and the eye disc does not reach its normal size and shape. Eyegone is a Pax-like protein (C. Desplan and H. Sun, personal communication to Hazelett, 1998). The expression patterns of dpp and wg were examined in early third instar eyg mutant discs. dpp expression is restricted to the posterior margin of eyg mutant discs, in contrast to its expression around the posterior and lateral margins of wild-type discs. On the contrary, wg expression was expanded, especially on the dorsal side of the disc, where it extends to the posterior margin. eyg thus acts to delimit the domains of dpp and wg expression; since it encodes a Pax-like transcription factor, it is possible that this regulation is direct. Since inhibition of the wg pathway at the posterior margin of eyg mutant discs is sufficient to allow photoreceptor formation, it is concluded that the misexpression of wg observed at the posterior of the eyg mutant discs is a major cause of the absence of photoreceptor development. As expected since it can overcome the effect of ectopic wg, activated Ras is also able to rescue photoreceptor differentiation in eyg mutant discs. In summary, these results show that wg inhibits normal photoreceptor differentiation in a manner independent of dpp expression or activation. The expression patterns of both dpp and wg, and perhaps their cross-regulatory interactions, are determined during early eye development by genes including eya and eyg (Hazelett, 1998).

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 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).

According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus, indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).

In contrast to ato broad expression just anterior to the furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential for uncommitted cells to acquire ommatidial cell fate or the ability to express ommatidial marker genes. Consistent with this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).

This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow. Uncommitted cells just posterior to the morphogenetic furrow are presumed to acquire ro expression potential at the earliest stage of the model (stage 1). In stage 2, R3/R4 precursors expressing ro acquire svp expression potential. svp expression in wild type R3/R4 precursors along with Ras1 activation-dependent svp misexpression in uncommitted cells is assumed to be not only positively regulated by the concerted action of Ras1 signaling and Dac and Eya but also negatively regulated by the protein product of the prepattern gene, lz. R1/R6 photoreceptors are recruited into ommatidia between stages 2 and 3. R1/R6 fate is previously shown specified by dual Bar homeobox genes, BarH1 and BarH2, whose expression is positively regulated by the cell-autonomous function of lz and svp. Consistent with this, in the putative R1/R6 arising area (around row 6), considerable svp expression occurs even in the presence of Lz. svp expression is regulated by Dac and Eya, so that normal Bar expression or R1/R6 fate eventually comes under the control of putative eye prepattern genes Lz, Dac, and Eya (Hayashi, 2001).

In stage 3, which may correspond to R7 and cone cell formation stages, pros is positively regulated through the concerted action of Ras1 signaling and prepattern gene lz (Hayashi, 2001).

In the developing Drosophila eye, differentiation of undetermined cells is triggered by Ras1 activation but their ultimate fate is determined by individual developmental potential. Presently available data suggest that developmental potential is important in the neurogenesis of vertebrates and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along the dorso-ventral axis in response to graded Sonic Hedgehog signals and this presages their future fates. Subdivision of originally equivalent neural progenitors through the action of prepattern genes may accordingly be a general strategy by which diversified cell types are produced through neurogenesis (Hayashi, 2001).

Identification of functional sine oculis motifs in the autoregulatory element of its own gene, in the eyeless enhancer and in the signalling gene hedgehog: Cooperation between So and Eya

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).

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).

The retinal determination gene eyes absent is regulated by the EGF receptor pathway throughout development in Drosophila

Members of the Eyes absent (Eya) protein family play important roles in tissue specification and patterning by serving as both transcriptional activators and protein tyrosine phosphatases. These activities are often carried out in the context of complexes containing members of the Six and/or Dach families of DNA binding proteins. eyes absent, the founding member of the Eya family is expressed dynamically within several embryonic, larval, and adult tissues of Drosophila. Loss-of-function mutations are known to result in disruptions of the embryonic head and central nervous system as well as the adult brain and visual system, including the compound eyes. In an effort to understand how eya is regulated during development, a genetic screen was carried out designed to identify genes that lie upstream of eya and govern its expression. This study identified a large number of putative regulators, including members of several signaling pathways. Of particular interest is the identification of both yan/anterior open and pointed, two members of the EGF Receptor (EGFR) signaling cascade. The EGFR pathway is known to regulate the activity of Eya through phosphorylation via MAPK. These findings suggest that this pathway is also used to influence eya transcriptional levels. Together these mechanisms provide a route for greater precision in regulating a factor that is critical for the formation of a wide range of diverse tissues (Salzer, 2010).

This report describes a genetic screen that identified factors that direct the expression of the retinal determination gene eyes absent to the developing embryonic head and eye imaginal disc. Putative regulators were identified by the loss or expansion of Eya protein distribution within the embryonic head of stage 9 loss-of-function mutants. The findings indicate multiple signaling cascades including Notch, Hedgehog, TGFβ, and the EGFR regulate eya expression. These results are consistent with previous studies identifying Hedgehog, Ras, and TGFβ as regulators of eya function in eye development. No mutations were recovered in any of known Wingless pathway members. This was slightly unexpected as Wnt signaling and eya are known to reciprocally regulate each other. This result could imply, however, that eya is regulated differently in diverse tissues (Salzer, 2010).

A screen similar to the one described in this study successfully identified the TGFβ pathway as an important upstream regulator of another retinal determination gene, dachshund. Of interest is the observation that the loss of TGFβ signaling has differential effects on eya and dac expression. In TGFβ mutant embryos ectopic dac expression was observed in cells of the visual primordium. However, eya expression remains unaffected in this tissue and is instead lost in the subsets of cells that give rise to the protocerebrum. These differential effects are interesting as eya and dac interact genetically within the retinal determination network. Therefore it seems that these regulatory relationships vary among different tissues. It also appears that the number of distinct signaling pathways that regulate eya expression outnumbers that of dac. This is unsurprising as the expression pattern of eya, when compared to dac, is considerably more dynamic, at least within the embryonic head (Salzer, 2010).

It was of particular interested to finding that mutations in spitz, argos, anterior open/yan and pointed, all members of the EGFR signaling pathway, altered the transcriptional pattern of eya. Previous work has demonstrated that the EGFR pathway post-translationally regulates Eya activity in the developing eye through phosphorylation via Ras/MAPK at two sites within the transactivation domain. Experiments in both flies and in insect cell culture indicate that phosphorylation augments, but is not absolutely essential, for either the transcriptional activation potential of Eya or for the induction of ectopic eyes in forced expression assays (Salzer, 2010).

These findings suggest that the EGFR pathway is also required to regulate eya transcription. This is consistent with findings that eya expression is lost in mago- clones, which reduce Ras signaling (Firth, 2009). Indeed, loss of aop/yan behind the morphogenetic furrow results in the higher levels of Eya and its facultative partner So. Both proteins are required for photoreceptor cell fate specification and maintenance. Elevated levels of Eya and So proteins in yan mutant clones are consistent with roles for Yan in suppressing photoreceptor cell fate during normal development. In yan clones, Eya protein levels are activated to significantly higher levels than that of So. One possible explanation for these results is that EGFR signaling may in fact regulate eya expression but not that of so. As EGFR signaling also regulates Eya activity, in a yan clone there may be a feedback loop that ultimately results in lowered levels of Eya phosphorylation. Reduced levels of the Eya phospho-protein, while still able to stimulate so transcription, may do so at a less efficient rate thereby leading to lower levels of ectopic So protein (Salzer, 2010).

Unexpectedly, it was found that dac, a putative downstream target of the So-Eya complex, is not up regulated in yan clones. Rather, dac expression is down-regulated when yan is removed. As So-Eya is thought to positively regulate dac expression this result is somewhat puzzling. The result does suggest that dac is regulated not only by the Eya-So complex but also by other mechanisms, possible through EGFR signaling and an intermediate repressor. The So-Eya-Dac subcircuit is under complex regulatory control. This study suggests that still greater complexity exists in the form of differential regulation by signal transduction cascades both at transcriptional and post-translational levels (Salzer, 2010).

Protein interactions

So and Eya 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 Dachshund and Eyes Absent proteins can physically interact through conserved domains, suggesting a molecular basis for the genetic synergy observed; it has been shown that a similar complex may function in mammals. The C-terminal portion of Eya interacts with Dac while the amino-terminal portion does not, suggesting that the C-terminal conserved domain of the Eya protein is contacting a portion of the Dac protein that is also conserved (Chen, 1997).

Drosophila Eyes absent function is positively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation: this regulation extends to developmental contexts independent of eye determination. In vivo genetic analyses, together with in vitro kinase assay results, demonstrate that Eya is a substrate for extracellular signal-regulated kinase, the MAPK acting downstream in the receptor tyrosine kinase (RTK) signaling pathway. Thus, phosphorylation of Eya appears to provide a direct regulatory link between the RTK/Ras/MAPK signaling cascade and the retinal determination gene network (Hsiao, 2001).

To address the possibility that Eya might be a direct downstream target of the RTK pathway, the Eya sequence was examined for potential MAPK phosphorylation consensus sites, defined as P-X-S/T-P (P, proline; X, any amino acid; S/T, serine or threonine). Investigations of alternative possibilities, including direct transcriptional regulation or protein-protein interactions between Yan and Eya, have yielded negative results to date. Two sites matching the consensus were found and will be referred to as 'MAPK sites.' Both MAPK sites are located ~80 residues upstream of the highly conserved 'Eya domain,' which has been shown to mediate interactions with So and Dac proteins. Examination of mammalian Eya protein sequences reveals similarly located MAPK sites in mouse and human Eya1, Eya2, and Eya4, suggesting that MAPK phosphorylation might be important for an evolutionarily conserved aspect of Eya function or regulation. Eya1 contains two MAPK sites, whereas Eya2 and Eya4 retain the second site but have a less stringent two amino acid consensus, S/T-P, at the first. No consensus MAPK sites were found in Eya3, although the less stringent two amino acid consensus is present at the second site (Hsiao, 2001).

To investigate the importance of the two MAPK phosphorylation sites with respect to Drosophila Eya function, the effects of mutating the phosphoacceptor residues were assessed in transgenic flies using an ectopic eye induction assay. First, site-directed mutagenesis was used to replace the serine residues in both sites with alanine (referred to as EyaS-A), effectively destroying the MAPK sites. Second, the serine residues were mutated to glutamic or aspartic acid (referred to as EyaS-D/E) to mimic the negative charge associated with phosphorylation. Transgenic lines expressing wild-type Eya (referred to as EyaWT), EyaS-A, and EyaS-D/E under control of the UAS promoter were used to induce tissue-specific expression. For each construct, ten independent lines were assayed, and the data from the strongest and weakest were discarded. For the remaining eight lines, ~300 progeny of the appropriate genotype were scored for each cross, and the percentage of flies exhibiting ectopic eye induction was calculated (Hsiao, 2001).

On average, expression of EyaWT induced ectopic eye formation in ~50% of the flies assayed. Most ectopic eyes were found on the head near the eye and around the base of the antenna. Patches of red eye pigment were also frequently induced on the wing hinges and underside of the metathoracic legs as well as occasionally on the wings, legs, and antennae. As expected when surveying a collection of independent transgenic insertions, positional effects resulted in a fairly broad distribution of phenotypic strengths. Thus, penetrance of ectopic eye induction ranged from almost 80% in the strongest line to <5% in the weakest. Strikingly, when the nonphosphorylatable EyaS-A transgene is expressed, the average incidence of ectopic eye formation dropped to ~20% as compared with the 50% observed with EyaWT. In addition, the size of the ectopic eye patches is noticeably reduced in EyaS-A. Thus, on average, mutation of the two MAPK sites to a nonphosphorylatable form reduces Eya's effectiveness at inducing ectopic eye formation (Hsiao, 2001).

In the converse experiment in which the MAPK sites were mutated so as to mimic the constitutively phosphorylated state (EyaS-D/E), the incidence of ectopic eye formation increased to an average value of ~80%. Furthermore, flies expressing EyaS-D/E exhibit an increase in the size and number of ectopic eye patches relative to flies expressing EyaWT. For example, in EyaWT lines, ectopic eyes usually form on only one side of the head, whereas in EyaS-D/E lines, ectopic eye induction often occurs bilaterally. Thus, mimicking the constitutively phosphorylated state dramatically enhances the efficacy of the Eya transgene in promoting ectopic eye formation. As with EyaS-A, EyaS-D/E lines exhibited no obvious changes in expression, stability, or subcellular distribution of the protein product that might provide alternate explanations for the apparent increase in activity (Hsiao, 2001).

To assess whether both MAPK sites are critical for Eya function, transgenes containing single MAPK site mutations were assayed for ability to promote ectopic eye formation. These transgenes are referred to as Eya1S-A, Eya2S-A, Eya1S-D, and Eya2S-E, where 1 and 2 refer to the specific MAPK site mutated. Expression of these transgenes produces intermediate results compared with the lines in which both MAPK sites were altered. For example, both Eya1S-A and Eya2S-A transgenes exhibited an average of ~30% ectopic eye induction, a value that represents an increase relative to EyaS-A but a decrease relative to EyaWT. Similarly, both Eya1S-D and Eya2S-E transgenes exhibited an intermediate value of ~65%, suggesting reduced activity relative to EyaS-D/E but increased activity relative to EyaWT. Together, these results suggest that both MAPK sites additively contribute to Eya function and regulation (Hsiao, 2001).

When interpreting the consequences of overexpressing a transgene, genetic determination of whether the observed phenotype reflects an antimorphic (dominant negative) or a hypermorphic (dominant activated) effect helps in assessing whether the particular transgene is functioning in a positive or negative manner relative to other regulators of the pathway. By genetically reducing the dose of the endogenous gene, it is possible to distinguish between the two possibilities (Hsiao, 2001).

To confirm that the ectopic eye induction associated with expression of the Eya transgenes reflects an increase in Eya activity, it was asked whether a 50% reduction in dosage of endogenous eya could suppress the phenotype. In all three cases (EyaWT, EyaS-A, and EyaS-D/E), flies heterozygous for a null eya allele showsweaker and less penetrant phenotypes. For both EyaWT and EyaS-A, a 50% reduction in ectopic eye formation is observed. Suppression is less pronounced with EyaS-D/E transgenes, consistent with the interpretation that EyaS-D/E is a hyperactivated protein (Hsiao, 2001).

These results, in conjunction with the finding that the EyaS-A and EyaS-D/E products are less and more efficient at promoting ectopic eye induction, respectively, suggest that Eya function is positively regulated via phosphorylation of these two MAPK sites in vivo. The fact that some activity is retained in the EyaS-A transgenes implies that MAPK phosphorylation does not provide a simple on/off switch, but rather serves to modulate the level of Eya activity (Hsiao, 2001).

The results obtained from overexpressing the EyaWT, EyaS-A, and EyaS-D/E transgenes implicate MAPK phosphorylation as positively regulating Eya activity. Drosophila, like mammals, has multiple MAPK family members, including ERK, that mediates RTK/Ras initiated signals, the Jun N-terminal Kinase (JNK), and the p38 stress-responsive MAPKs. Less is known about the upstream signaling mechanisms that activate JNK and p38 MAPKs, although both families respond to stress and may exhibit some functional redundancy. Although isolation of Eya in an RTK pathway-based genetic screen made ERK the best candidate, other MAPK family members could also potentially phosphorylate Eya. Currently, specific loss-of-function mutations are available for only two members of the MAPK family in Drosophila: ERK and JNK. The gene rolled encodes ERK, whereas the gene basket encodes JNK. Two stress-responsive MAPKs, p38a and p38b, have been cloned and characterized in overexpression assays, although specific mutations have not yet been reported (Hsiao, 2001).

To address whether phosphorylation of Eya by MAPK is physiologically relevant, it was asked whether genetically reducing the dose of either ERK (rolled) or JNK (basket) could suppress the phenotypes associated with Eya overexpression. rolled mutations dramatically suppresses EyaWT phenotypes, whereas basket mutations have no effect. Strong suppression of EyaWT phenotypes was also obtained with mutations in the Epidermal growth factor receptor (Egfr), suggesting that activity of the canonical RTK/Ras/MAPK cascade participates in modulating Eya activity. Together, these results suggest that in vivo, ERK, the MAPK responsive to RTK-initiated signals, but not JNK, positively regulates Eya activity. A double mutant between rolled and basket suppresses EyaWT at the same level as rolled alone, suggesting there are no synergistic effects between these two kinases. In a related experiment, it was found that coexpressing EyaWT and an activated allele of ERK enhances the penetrance and severity of the phenotypes, whereas coexpression of EyaWT and JNK do not (Hsiao, 2001).

Interestingly, a reduction in rolled dosage is also able to suppress the phenotypes associated with EyaS-A transgenes. Because the two MAPK sites are effectively destroyed in this construct, the suppression is unlikely to reflect a direct interaction between ERK and the EyaS-A product. Two possible interpretations could explain this result: (1) additional sites in Eya could be phosphorylated by ERK, apart from the two MAPK consensus sites identified; (2) the suppression could simply reflect downregulation of endogenous Eya. The fact that reduction in rolled dosage suppresses to a greater extent than reduction in eya dosage is consistent with this interpretation, assuming that the pool of endogenous ERK is limiting. In this case, then the amount of ERK produced in a heterozygous rolled background would be insufficient to activate even 50% of the endogenous Eya, and thus, even stronger suppression would be obtained relative to that seen with a 50% reduction in endogenous eya (Hsiao, 2001).

Consideration of the genetic interaction data obtained with the EyaS-D/E transgenes increases the likelihood of the second scenario. A 2-fold reduction of either endogenous eya or endogenous rolled led to a comparable ~15% reduction in penetrance of ectopic eye induction in the EyaS-D/E background. If Eya function were regulated via MAPK phosphorylation at additional sites, reducing the dose of rolled should have a greater impact on the phenotype than reducing the dose of eya, even in the hyper-activated EyaS-D/E background. Thus, the explanation is favored that suppression of EyaS-A and EyaS-D/E upon reduction of endogenous ERK is primarily attributable to the dose sensitivity of the phenotype relative to endogenous Eya activity. The fact that suppression of EyaS-D/E is less striking than that observed in either the EyaWT or the EyaS-A backgrounds is consistent with the hypermorphic nature of the EyaS-D/E product (Hsiao, 2001).

To complement the in vivo genetic experiments and to determine whether the interaction between Eya and MAPK is direct, kinase assays were performed to assess which MAPK phosphorylates Eya preferentially in vitro. Activated ERK or JNK were immunoprecipitated from Drosophila S2 cultured cells transfected with appropriate cDNA constructs. Both kinases were active, as demonstrated by their ability to phosphorylate myelin basic protein and GST-Yan. GST-EyaWT and GST-EyaS-A fusion proteins were generated and tested for the ability to serve as ERK or JNK substrates. Consistent with the in vivo genetic results, ERK phosphorylates GST-EyaWT, whereas JNK, although clearly active, does not (Hsiao, 2001).

The p38a and p38b MAPKs were also tested for ability to phosphorylate Eya using the in vitro kinase assay. Both p38a and p38b phosphorylated GST-EyaWT at levels comparable to those obtained with ERK. These results suggest a possible role for the p38 MAPKs in activation of Eya, although in vivo validation of these predictions must await the isolation of specific mutations in p38a and p38b. However, given the recent report suggesting that p38b MAPK functions downstream of dpp signaling, it is tempting to speculate that the kinase assay results may provide a mechanistic basis underlying the genetic synergy that has been demonstrated between dpp and the retinal determination genes (Hsiao, 2001).

Over the course of the experiments looking at frequency of ectopic eye induction, several phenotypes associated with Eya overexpression were noticed that had not been previously reported. Specifically, Eya overexpression results in wing defects, increased number of scutellar macrochaetae, and problems with thoracic closure. Additional phenotypes, including rough eyes and arista to leg transformations, were observed at low penetrance and have not been extensively characterized (Hsiao, 2001).

Although eya loss-of-function phenotypes have not been reported in adult tissues other than the eye and germ line, it was found that all of the phenotypes associated with Eya overexpression can be suppressed by a 50% reduction in endogenous eya. This argues strongly that eya is expressed and could play a role in the normal development of these tissues. Furthermore, similarly strong suppression is achieved upon reducing the dose of endogenous dac or so, suggesting that part or all of retinal determination gene network may be redeployed in developmental contexts independent of eye formation (Hsiao, 2001).

Genetic tests to determine which MAPK might be responsible for modulating Eya activity in these contexts were performed as described for the ectopic eye phenotype. Because the thoracic closure and macrochaetae phenotypes were more variable and therefore less conducive to quantitative analyses, focus was placed on Eya regulation in the wing. A reduction in rolled dosage strongly suppresses whereas a reduction in basket dosage has little effect on the observed wing phenotypes. Conversely, coexpression of EyaWT and activated ERK enhances the severity of the wing defects, whereas coexpression of JNK has little effect. Because expression of activated ERK alone results in ectopic wing vein formation, it is formally possible that the enhancement is additive rather than synergistic. However, within the same wing, the two phenotypes are easily distinguished, and the best interpretation is that the Eya-associated defects are enhanced whereas the ERK-associated ones are not. As was found in the ectopic eye formation assay, mutations in Egfr also suppressed the wing phenotypes. Thus, in the contexts of both eye and wing development, Eya activity appears to be modulated by ERK, the MAPK functioning downstream of the RTK signaling pathway (Hsiao, 2001).

These results suggest RTK signaling plays dual and opposing roles during eye development. Previous work has indicated that an early, MAPK-independent aspect of RTK signaling antagonizes the decision to become an eye. The current study shows that a MAPK-dependent signal positively promotes eye morphogenesis later in development by directly modulating Eya activity. The fact that the same pathway can exert such apparently opposing effects is not unusual; depending on the specific context, the same signal may be interpreted in strikingly different ways (Hsiao, 2001 and references therein).

Groucho interaction with Engrailed homology 1 (eh1) proteins

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).

The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).

Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).

Regulation of the retinal determination gene dachshund in the embryonic head and developing eye

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).

Interactions with the abelson tyrosine kinase reveal compartmentalization of eyes absent function between nucleus and cytoplasm

Eyes absent (Eya), named for its role in Drosophila eye development but broadly conserved in metazoa, possesses dual functions as a transcriptional coactivator and protein tyrosine phosphatase. Although Eya's transcriptional activity has been extensively characterized, the physiological requirements for its phosphatase activity remain obscure. This study provides insight into Eya's participation in phosphotyrosine-mediated signaling networks by demonstrating cooperative interactions between Eya and the Abelson (Abl) tyrosine kinase during development of the Drosophila larval visual system. Mechanistically, Abl-mediated phosphorylation recruits Eya to the cytoplasm, where in vivo studies reveal a requirement for its phosphatase function. Thus, a model is proposed in which, in addition to its role as a transcription factor, Eya functions as a cytoplasmic protein tyrosine phosphatase (Xiong, 2009).

This analysis of the subcellular compartmentalization of Eya function has revealed a requirement for Eya activity in the cytoplasm. Specifically, although nuclearly restricted NLS-Eya, with an inserted nuclear localization sequence, appears to be fully competent as a coactivator, as judged by cultured cell transcriptional reporter assays, it exhibits a reduced ability to induce eye tissue in either wild-type or eya loss-of-function backgrounds. Coexpression of cytoplasmically restricted Myr-Eya (containing a Src myristoylation tag) restores a wild-type level of eye inducing activity to the NLS-Eya background, supporting the interpretation that NLS-Eya is fully competent with respect to transcription, but cannot perform the essential function normally provided by cytoplasmic Eya. Eya phosphatase activity appears to contribute to cytosolic function, since phosphatase-dead versions of cytoplasmically restricted Eya transgenes fail to complement the NLS-Eya background effectively. Thus, it is proposed that whereas regulation of gene expression by the core retinal determination (RD) network relies primarily on nuclear Eya function, other signaling events important for retinal development may rely on transcription-independent functions of the cytoplasmic Eya phosphatase (Xiong, 2009).

Mechanistically, it is proposed that Eya traffics dynamically between nuclear and cytoplasmic compartments, with its final localization determined by its phosphorylation state and interactions with specific signaling partners. Thus, in contexts in which Abl signaling is activated, Abl-mediated phosphorylation may provide a cytoplasmic retention signal that targets Eya to its appropriate site of action, presumably through interactions with specific phosphotyrosine-binding proteins. Autocatalytic Eya phosphatase activity would play a critical positive role with respect to overall Eya function by returning Eya to the nucleus to prevent depletion of the nuclear pool needed to carry out essential transcriptional programs. Although cytosolic Eya substrates have not yet been identified, the fact that phosphatase-dead cytoplasmically restricted Eya was less active than the wild-type version in an assay in which dynamic shuttling between nuclear and cytoplasmic compartments was not relevant suggests that Eya-mediated dephosphorylation of substrates other than itself is likely important (Xiong, 2009).

Although Eya has been primarily characterized as a nuclear protein, two previous observations are consistent with the proposed model of extranuclear function: (1) in mammalian cultured cells, Eya nuclear localization and/or retention requires the presence of its binding partner Six, such that in its absence Eya localizes to the cytosol; (2) protein-protein interactions with several membrane-associated and cytoplasmic proteins have been demonstrated in two-hybrid screens, although only one interaction has been further investigated. In this example, interactions between Eya and the G protein Gαi can recruit Eya to the cytoplasm of cultured cells, and a balance between binding to G proteins and Six has been proposed to regulate Eya distribution and function (Xiong, 2009 and references therein).

To what aspects of eye development might cytosolic Eya activity contribute? Although identification of Eya substrates and elucidation of the specific signaling events regulated by cytoplasmic Eya activity will be required to answer this question definitively, several intriguing models are worth considering. (1) The ectopic eye induction and genetic rescue assays used to characterize the complementation between Myr-Eya and NLS-Eya transgenes imply a requirement for extranuclear Eya in retinal specification. In considering this context, it is important to note that whereas a great deal is understood about how transcriptional hierarchies such as the RD network drive retinal induction, much less is known about how specific differentiation programs are coordinated with the morphogenetic events that pattern the tissue. In Drosophila, specification of retinal fates is immediately preceded by adhesive and morphological changes in and posterior to the morphogenetic furrow. Although phosphotyrosine signaling at the morphogenetic furrow has not been extensively studied, its importance to cell adhesion and epithelial morphogenesis in other contexts is well documented. For example, recent work studying the invagination of the ventral furrow during Drosophila gastrulation demonstrated that Abl signaling acting in parallel to the Rho activator RhoGEF2 regulates actin organization to drive apical cell constriction. Given the importance of cell constriction in the retinal furrow, it will be interesting to investigate whether similar signaling mechanisms operate in this context and whether cytoplasmic Eya phosphatase activity is involved. Encouragingly, loss of eya impairs morphogenetic furrow propagation, suggesting that investigation of defects in epithelial remodeling and reorganization of cell-cell contacts at the furrow in eya mutants could be fruitful (Xiong, 2009).

(2) Another possibility is that cytoplasmic Eya phosphatase function might provide critical feedback regulation on other signaling pathways during retinal specification. Indeed, a complex web of interactions between multiple signaling networks including the Wingless, Notch, Hedgehog, and EGFR pathways has been shown to be critical for RD network function and retinal induction. Thus, if cytosolic Eya phosphatase activity were absent or mislocalized, the resulting signaling imbalances could potentially compromise eye specification and development (Xiong, 2009).

(3) Finally, cytoplasmic Eya function could be important for neuronal morphogenesis, perhaps through involvement in Abl-mediated signaling events. Because Abl signaling has not yet been explored in the retina, determining which downstream branches of the pathway operate in this developmental context will be important for elucidating the molecular and cellular defects underlying the phenotypes and interactions that have been reported. For example, the photoreceptor axon targeting defects observed in eya or abl mutants, or upon Myr-EyaWT expression, could reflect impaired receiving or processing of attractive signals from either brain cells or adjacent retinal neurons, weakening of repulsive signaling between axons, or strengthening of adhesive properties between the axons such that they fail to spread properly as they exit the optic stalk (Xiong, 2009).

In considering the mechanistic possibilities whereby Eya might interact with the Abl signaling network, it is important to reiterate that although genetic analyses indicate eya and abl function cooperatively, the two genes encode proteins with opposing catalytic functions. Thus, a simple relationship whereby Eya dephosphorylates Abl or its substrates is unlikely to offer a suitable explanation since this would most likely be reflected as antagonism rather than synergy. Instead, Abl phosphorylation and recruitment of Eya to the cytoplasm may facilitate formation of protein complexes important for Abl signaling and/or promote interactions with components of other phosphotyrosine signaling pathways, which together would target Eya phosphatase activity toward appropriate substrates. Finally, these results do not preclude Eya's nuclear transcriptional activities from also contributing to Abl signaling; thus, it will be important to investigate further the complex spatiotemporal requirements for Eya, other members of the RD network, and the Abl signaling pathway during retinal development (Xiong, 2009).

eyes absent: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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