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Genetics. Jan 2010; 184(1): 185–197.
PMCID: PMC2815915

The Retinal Determination Gene eyes absent Is Regulated by the EGF Receptor Pathway Throughout Development in Drosophila

Abstract

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 the fruit fly, Drosophila melanogaster. 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, we have carried out a genetic screen designed to identify genes that lie upstream of eya and govern its expression. We have 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. Our 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.

IN Drosophila, an evolutionarily conserved regulatory network executes early decisions within the retina. This network includes a dozen known nuclear proteins that serve as DNA-binding proteins, transcriptional co-activators, phosphatases and kinases (Kumar 2009). Much effort into understanding the genetic, molecular, and biochemical mechanisms that underlie the function of this network has revealed that it does not function as a simple linear cascade with a unidirectional flow of information. Rather, the network is characterized by a meshwork of interactions that include numerous feedback loops and closed auto regulatory circuits (Kumar 2009). Additionally, several signaling transduction pathways function reiteratively within the network (Chen et al. 1999; Baonza and Freeman 2001; Kurata et al. 2000; Hsiao et al. 2001; Kumar and Moses 2001b,c; Baonza and Freeman 2002; Voas and Rebay 2004). Complicating our understanding of this network is that all of the interactions described to date do not necessarily occur uniformly throughout the eye. Instead, the functioning of the network seems to be influenced by spatial and temporal considerations (Salzer and Kumar 2009).

The eyes absent (eya) gene plays a central role within the retinal determination network. It encodes a transcriptional co-activator that also serves as a protein tyrosine phosphatase (Li et al. 2003; Rayapureddi et al. 2003; Silver et al. 2003; Tootle et al. 2003). Like the other members of the network, eya is expressed and functions within multiple tissues during development (Leiserson et al. 1998; Bonini et al. 1993, 1998; Bai and Montell 2002; Fabrizio et al. 2003). Null mutants die during embryogenesis while mutations within an eye specific enhancer lead to viable animals completely lacking the compound eye (Bonini et al. 1993, 1998; Leiserson et al. 1998; Bui et al. 2000a,b; Zimmerman et al. 2000). In contrast, forced expression of eya in several nonretinal tissues is sufficient to induce ectopic eye formation (Bonini et al. 1997).

Eya and its mammalian homologs influence development through two distinct biochemical mechanisms. First, they serve as transcriptional activators within a complex that often includes members of the Six and Dach families of homeobox DNA-binding proteins (Chen et al. 1997a; Pignoni et al. 1997; Xu et al. 1997; Ohto et al. 1999; Ikeda et al. 2002; Silver et al. 2003). As Six proteins appear to be lacking in strong intrinsic activation properties, Eya proteins are critical to promoting the expression of Six-Eya targets (Pignoni et al. 1997; Jemc and Rebay 2007a). Second, Eya proteins have been shown to possess tyrosine phosphatase activity (Rayapureddi et al. 2003; Tootle et al. 2003; Rebay et al. 2005). This activity appears to be required for Eya to serve as a transcriptional activator, as mutations that reduce the phosphatase activity of Eya proteins reduce the ability of the Six-Eya complex to interact with DNA (Li et al. 2003; Mutsuddi et al. 2005; Jemc and Rebay 2007b). More recently, Eya phosphatase activity has been shown to be required for appropriate embryonic CNS axonogenesis as well as photoreceptor axon guidance in Drosophila (Xiong et al. 2009). These recent findings, taken with work previously completed in mammalian cell culture, suggest that Eya had distinct developmental responsibilities in both the cytoplasm and the nucleus (Fan et al. 2000; Embry et al. 2004; Xiong et al. 2009).

The wide-ranging expression patterns of eya and the ability of Eya protein to function in both nuclear and cytoplasmic compartments suggests that its regulation may be complicated and occur at many levels. Indeed, Eya activity is modulated post-translationally via phosphorylation by EGFR/MAPK signaling (Hsiao et al. 2001) while its subcellular localization is regulated via interactions with select G-α subunits in mammalian cell culture (Fan et al. 2000; Embry et al. 2004). We set out to identify genes that lie genetically upstream of eya and regulate its expression. We conducted a screen for mutants that alter the distribution pattern of Eya protein in the developing embryonic head. From this effort we isolated a number of putative upstream transcriptional regulators including representatives from several signaling pathways. In particular, we demonstrate that the EGF Receptor signaling pathway regulates the expression of eya through the Ets transcription factors pointed (pnt) and yan/anterior open. We also describe the putative regulatory relationship between this signaling pathway and two other retinal determination genes, sine oculis (so) and dachshund (dac).

MATERIALS AND METHODS

Fly stocks and genetic screen:

The Bloomington Drosophila Deficiency Kit was used to initially interrogate the genome for regions containing positive and negative regulators of eya expression. We collected stage 9 embryos homozygous for each chromosomal deletion within the kit and stained them with an antibody that recognizes the Eya protein. These deletions provide >95% coverage of the Drosophila genome. The embryos were assayed for changes in the eya expression pattern. As a secondary screen we repeated this analysis with single gene disruption mutations the lie within the subset of deficiencies that altered eya expression. Eya protein distribution was altered in the following mutant alleles: yan1, argosW11, bib1, cact1, CG1455d08265, CG3353UY1730, da1, dac1, dl4, dppH48, dvek065515, Egfrk05115, emc1, exu1, lab14, l(1)G0344G0344, l(1)G01290G0129, l(1)G0145G0145, l(2)k09221k09221, l(2)k07433e00176, l(2)k02107ak02107a, l(2)k05713k05713, l(2)k0711bk0711b, l(2)k07237k07237, l(2)k13704k13704, l(3)k65ACfD54, Mad1-2, Me65d, mtsXE-2258, neur11, OceWC1, osa2, phlG0475, scw5, sec1301031, pntD88, ptc7, put135, so3, Sosk05224, spi1, srw1, stc05441, tkv7, tldB4, tokG3, tsg4, twi1, zen2. A second mutant allele of each gene was also shown to have altered eya expression patterns (a listing of these mutant alleles is available upon request).

The following GAL4 lines were used in this study in forced expression assays: ey-GAL4, dpp-GAL4, act5C-GAL4, GMR-GAL4. The following UAS lines were used in this study: UAS-yan, UAS-aos, UAS-cact, UAS-da, UAS-dac, UAS-dl, UAS-dpp, UAS-dve, UAS-Egfr, UAS-emc, UAS-exu, UAS-ey, UAS-eya, UAS-eyg,, UAS-lab, UAS-Mad, UAS-mts, UAS-optix, UAS-osa, UAS-ptc, UAS-pnt P1, UAS-Pnt P2, UAS-put, UAS-Ras UAS-scw, UAS-so, UAS-spi, UAS-tkv, UAS-tld, UAS-toe, UAS-toy, UAS-tsg, UAS-twi, UAS-zen. The following stocks were used to generate mutant retinal mosaic clones: yweyflp; FRT42D Ubi-GFP/FRT42D aop1 and yweflp; FRT82B Ubi-GFP/FRT82B pntD88. A pnt-lacZ line was used to monitor pnt transcription in embryos and eye discs. All experiments were conducted at 25°.

Reagents and microscopy:

The following reagents were used in this study: mouse α-Dac (1:5), mouse α-Eya (1:5), guinea pig α-So (1:500, gift of Ilaria Rebay), rat α-Elav (1:100), mouse α-β Galactosidase, donkey α-mouse FITC (1:100), goat α-mouse Biotin (1:100), Streptavidin HRP (1:100), donkey α-rat FITC (1:100), goat α-guinea pig FITC (1:100), donkey α-rat Cy5 (1:100), donkey α-mouse Cy5 (1:100), phalloidin-TRITC (1:1000; Molecular Probes) and DAB Detection Kit (Pierce). All primary antibodies (with the exception of α-So) are from the Developmental Studies Hybridoma Bank and all secondary antibodies are from the Jackson Laboratories. Embryos, imaginal discs and adult eyes were prepared for light, fluorescent and scanning electron microscopy as essentially described in Anderson et al. (2006).

RESULTS

A screen for embryonic regulators of eya identifies four classes of regulators:

The expression of the eyes absent (eya) gene, like all members of the eye specification network, is not restricted to the developing retinal epithelium but extends to several non-retinal tissues. Within the developing embryonic head (at stage 9), Eya protein is distributed within the optic lobes (ol), visual primordium (vp), mid-dorsal head (mdh), protocerebrum (pc), procephalic lobes (pl), and clypeolabrum (cl) (Bonini et al. 1998) (Figure 1, A and B). In an effort to better understand how this dynamic expression pattern is achieved, we executed a genetic screen designed to identify putative transcriptional regulators of eya (Figure 1C).

Figure 1.
Distribution of Eya within the developing embryonic head of Drosophila (A and B) Dorsal and lateral views respectively of wild type stage 9 embryos stained with an antibody that recognizes Eya. (C) A schematic drawing describing the genetic screen the ...

Initially, we systematically screened 235 deficiency stocks by collecting and staining stage 9 embryos with an anti-Eya antibody and looking for changes in Eya distribution within the embryonic head (supporting information, Table S1). Throughout the screen we eliminated candidates that demonstrated early embryonic lethality or had gross defects in head morphology. Deficiencies that exhibited visibly altered eya expression, 57 in total, where selected for further analysis via smaller deficiencies and single gene disruptions within the cytologically mapped breakpoints. In this second phase of the screen we tested 173 smaller deficiencies (100 were positive for changes in eya) and 316 single gene disruption stocks (53 were positive for altered eya expression) to refine our genetic maps (Table S2). As members of well-known signaling pathways emerged, we were able to select additional members of each pathway to identify genes that may have been missed in our initial screen (3 were positive for changes in eya pattern). We reasoned that these genes may have been passed over (during the screening of deficiency stocks) because of early lethality or catastrophic developmental defects caused by the loss of large amounts of genetic material. In all, we were able to identify a total of 56 putative regulators of eya in the embryonic head, many of which have phenotypes (change in eya expression) that very closely resemble those seen within the larger deficiencies (Table 1). This approach allowed us to rapidly scan the genome and revealed loci of interest for more detailed phenotypic analysis.

TABLE 1
Putative regulators of eyes absent

Alleles identified in this screen can be placed into four broad categories, which we arbitrarily refer to as Class I–IV mutants (Table 1, Figure 2). It should be noted that while each group is characterized by a primary effect on Eya protein distribution, in some cases secondary phenotypes that may be common to mutants in multiple group are also present. Class I mutants are characterized by the distribution of Eya protein throughout the anterior-most portion of the head (Figure 2, A–D, arrows). Interestingly, in these mutants the entire head does not express eya; rather the ectopic expression is restricted to the anterior third to half of the head. In several mutants such as CG11455, l(3)65ACf and l(2)k07433 this pattern is accompanied by an expansion of eya expression (up to 10 cell diameters) within the vp (Figure 2, B–D, arrowhead). This is in contrast to the visual primordium of wild type embryos in which eya expression spans a width of only 2-3 cells (Figure 1, A and B). Additional defects such as a reduction of eya expression in the pc and ol can be seen in a subset of Class I mutants (Figure 2, C and D).

Figure 2.
Regulation of eya within the developing head of stage 9 embryos (A–P) Dorsal views of mutant stage 9 embryos stained with an antibody that recognizes Eya. Genotypes are listed in the bottom right corner of each panel. Arrows and arrowheads denote ...

Mutants within Class II are grouped together based on the strong reduction in eya within the vp and/or ol (Figure 2, E and H, arrowheads). In some mutants, such as extra macrochaetae (emc) and sec13, Eya is completely lost in both regions (Figure 2, E and F, arrows). However in other mutants, such as Mothers against dpp (Mad) and dorsal (dl), the loss of Eya protein is mainly confined to the ol and a portion of the vp (Figure 2, F and G, arrowheads). These two mutants differ from emc and sec13 in that Eya is still present in the mdh. There are additional defects in the more anterior expression domains; however these changes are variable among the mutants within this class.

Class III mutants share a reduction in eya within the pc and, to a lesser degree, diminished expression within the ol (Figure 2, I–L). For some of the members of this class, occasional embryos exhibiting Class II Eya patterns were observed. We attribute this phenotypic overlap between Class II and III to genes that fall in the same signaling pathways and may have different relationships with eya in different regions of the head (Table 1). Regardless, the majority of embryos for each Class III genotype exhibited Class III alterations to normal Eya protein distribution. Embryos mutant for twisted gastrulation (tsg), spitz (spi), dachshund (dac), and daughterless (da) exhibit similar changes in Eya distribution. In each mutant, eya is completely lost in regions just anterior to the vp and within the ol (Figure 2, I–L, arrows). In contrast, there is an increase in the number of rows of Eya positive cells within the vp. This is especially notable in da mutants (Figure 2L, arrowhead). The broadening of eya expression in da mutants is similar to that seen in class I mutants. However, da remains in its present grouping due to the relatively normal Eya expression in the anterior-most portions of the head (compare Figure 2L to 2, B–D).

Class IV represents mutants that we were not able to place in any of the three previously described classes. Changes in eya expression in members of this group vary from nearly global loss to selective reduction of eya within a limited number of cells. We have chosen four representatives from this group for discussion here (Figure 2, M–P); we will address another Class IV gene, aop, in more detail later (see below). The first example, neuralized (neur), is member of the Notch signaling pathway. In these mutants we observe a loss of Eya protein in regions of the vp that connect the ol to the mdh (Figure 2M, arrows). Additionally, neur mutants display a narrowing of the wild type eya expression pattern within the pc (Figure 2M, arrowhead). This is contrasted in defective proventriculus (dve) mutants where eya expression is lost in the mdh, the posterior portion of the pc, and the ol leaving only two narrow strips of the visual primordium to express eya (Figure 2N arrows). In addition, dve mutants show a slight broadening of eya across the anterior part of the head including the anterior portion of the pc (Figure 2N, arrowhead). Similarly, exuperantia (exu) mutants have a wider patch of eya expressing cells in the anterior portion of the head and thickening of the vp (Figure 2O, arrowhead, arrow). The vp thickening is a common characteristic of Class I mutants, however exu mutants do not display the same increase in Eya protein distribution in the anterior-most regions of the head that we see in all Class I members (compare Figure 2O to 2B,C). Finally, sine oculis (so) mutants exhibit some characteristics of neur and dve mutants: eya expression is lost in the ol and in subsets of cells of the pc. Levels of Eya protein are also dramatically reduced in the vp, which is a common feature of Class II mutants (Figure 2P, arrow & arrowhead).

Interestingly, two RD genes, sine oculis (so) and dachshund (dac), emerged from our screen as embryonic eya regulators (Figure 2, K and P). With respect to each other, so and eya are thought to reside at the same level within the RD hierarchy. In the developing retina they have both been shown to be directly activated by the transcription factor, ey (Halder et al. 1998; Niimi et al. 1999; Ostrin et al. 2006) and So and Eya protein products form a biochemical complex whose function is thought to be crucial for promoting eye formation through the activation of downstream target genes (Pignoni et al. 1997). One of these targets is dac, a gene that occupies the lowest known position within the cascade. Additionally, Dac and Eya themselves have been shown to form a biochemical complex in vitro though more recent work suggests that formation of this complex might not be necessary for eye development (Chen et al. 1997a; Tavsanli et al. 2004). In the eye disc, the So-Eya-Dac subcircuit is flexible and the regulatory relationships change in a position dependent manner (Salzer and Kumar 2009). In most regions of the eye imaginal disc So and Eya participate in their canonical positive regulatory role with respect to dac. However in the very posterior regions of the tissue this relationship changes and So represses dac through partnership with a co-repressor. And at the margin of the eye disc there is feedback stability among all three members of this RD subcircuit (Salzer and Kumar 2009). Interestingly, based on our data in the embryo, it appears that a feedback loop exists between dac and eya in this developmental context as well as in the margin of the eye disc. It should be noted that Eya and Dac protein distribution in stage 9 embryos is largely non-overlapping (Kumar and Moses 2001b) suggesting that part of the change we see in eya expression in dac mutants may be non-autonomous or may occur indirectly. At earlier stages in embryonic development (~5hr AEL), so and dac expression overlap in the ol, thus it is possible that at this stage dac positively regulates so and so, in turn, regulates eya (Kumar and Moses 2001b).

Expression of putative regulators is sufficient to alter Eya protein distribution:

Using the UAS/GAL4 system we forcibly expressed a subset of the genes that are listed in Table 1 (marked with an asterisk) throughout the embryo in an attempt to determine if these genes, on their own, are sufficient, to influence eya expression. The genes were chosen based on the availability of extant UAS-driven transgenes. Several genes, such as dl, proved necessary for normal eya expression based on mutant analysis, but were incapable of altering Eya protein distribution in this forced expression assay (Table 1; Figure 2F, ,3A).3A). There are a few possible explanations for this result; it may be that these putative regulators require co-factors that have not been provided in the assay. Alternatively, they may not have been expressed at high enough levels to see a change in Eya distribution. Another possibility could be that there may be other factors at play within a signaling pathway that limit the function of the overexpressed gene and its encoded protein. And finally, it is possible that the connection between the putative regulator and eya is so indirect that overexpression of the regulator is incapable of having a direct effect on the transcription of eya.

Figure 3.
Change in Eya distribution in response to the expression of putative regulators (A–D) Dorsal views of stage 9 embryos in which the Act5C-GAL4 driver expresses individual regulators throughout the embryo. Genotypes are listed in the bottom right ...

On the other hand, many genes are, in fact, capable of altering eya expression. For instance, down regulation of the TGFβ signaling pathway leads to a strong loss of eya expression in the pc while increased levels has the opposite effect: Eya protein distribution is expanded within the pc and vp of animals overexpressing dpp transgenes (Figure 2I, ,3B,3B, arrows and arrowheads). Expression of a subset of genes such as dac is sufficient to completely abolish or drastically reduce eya expression from the embryo (Figure 3C). There are also occasional instances in which we cannot determine the effect that global overexpression of the regulators has on Eya protein distribution because forced expression leads to early embryonic lethality. This was the case when we expressed so throughout the developing embryo. In this case the embryo failed to even undergo germband extension (Figure 3D).

Effects of forced expression on Eya protein distribution in the developing eye:

We were interested in determining if putative eya regulators could also function to govern eya expression in the retina. Normally eya is expressed in a broad stripe ahead of the morphogenetic furrow (approximately 20 cell diameters wide), in all developing photoreceptor and cone cells, all undifferentiated cells behind the morphogenetic furrow, and within the developing ocelli (Figure 4A, furrow marked with arrowhead and dashed line; (Bonini et al. 1993). An enhancer of the ey gene directs expression ahead of the furrow to a broader swathe of cells when compared to the known eya enhancer (data not shown). Using the ey-GAL4 driver, we expressed a subset of genes from Table 1 in all cells ahead of the furrow and then used an antibody to assay the effect on Eya protein distribution.

Figure 4.
Overexpression of putative eya regulators in cells ahead of the morphogenetic furrow. (A) Scanning electron micrograph of a wild type eye and immunofluorescence image of a wild type eye disc stained with an antibody that recognizes Eya protein. (B–F) ...

We first focused on genes known to function during retinal determination. Forced expression of decapentaplegic (dpp), a member of the TGFβ superfamily, activates eya expression in all cells ahead of the furrow and appears to initiate ectopic eye formation at the anterior margin of the eye field. Often the end result is the production of two compound eyes being generated from a single retinal field (Figure 4B). This result is consistent with previous work that demonstrated that dpp functions within the retinal determination network to promote eye specification and furrow initiation (Pignoni and Zipursky 1997; Shen and Mardon 1997; Hazelett et al. 1998). It is also consistent with the effects that we see in the embryo in which activation of TGFβ signaling in the embryonic head leads to ectopic eya expression in the pc and the vp (Figure 3B). Similarly, expression of dac ahead of the furrow in the developing eye is also capable of inducing eya expression (Figure 4C). This in contrast to what we observe in the embryo where over-expression of dac results in the strong repression of eya (Figure 3C).

Forced expression of so did not have the same effect on eya expression. Instead of activating eya transcription and/or generating a larger eye field, the ey-GAL4, UAS-so eyes are smaller with little to no effect on the pattern of Eya protein distribution ahead of the furrow (Figure 4D). We then looked at how the forced expression of two genes from Table 1 [dve and osa] influence eya expression in the retina. These genes were chosen since they are not previously known to function in eye specification. Expression of dve in the eye disc leads to increases in both cell proliferation and eya expression (Figure 4E). In contrast, expression of dve within the embryonic head results in the downregulation of eya (data not shown). This represents another example of the same gene having distinctly opposite regulatory effects on its target depending on the stage of development. Expression of osa, which is known to be involved in furrow initiation and retinal differentiation does alter the structure of the eye but does not have a direct effect on eya expression (Figure 4F).

Since Eya is normally present in all cells behind the furrow, we also expressed each putative regulator under the control of GMR-GAL4, which drives expression in these cells. In cases where GMR-GAL4/UAS-putative regulator adults demonstrated aberrant retinal morphologies we dissected and stained eye discs and looked for changes in Eya expression. While several putative regulators caused eye phenotypes ranging from mildly rough to severely glazed (Figure S1), with the exception of yan/anterior open (see below), we observed no significant changes in Eya protein distribution in third instar eye discs (data not shown).

The EGF Receptor pathway regulates eya expression:

In our screen we identified members of the TGFβ, Notch, and EGFR pathways, a result that connects these signaling cascades in the regulation of eya expression within the embryonic head (Figure 2, Table 1). Previous work has also implicated these pathways in regulating both eya and several other members of the retinal determination network (Chen et al. 1999; Curtiss and Mlodzik 2000; Kurata et al. 2000; Hsiao et al. 2001; Kumar and Moses 2001a; Firth and Baker 2009). Because the EGF Receptor pathway regulates Eya activity in the retina by phosphorylation via the downstream cytoplasmic effector protein MAPK (Hsiao et al. 2001; Firth and Baker 2009), we chose to take a closer look at yan/anterior open (aop) and pointed (pnt), two Ets transcription factors that lie downstream of the EGF Receptor (O'Neill et al. 1994; Rebay and Rubin 1995).

Homozygous mutant aop and pnt embryos each exhibit striking changes to the eya expression pattern in the embryo. In aop mutants we see changes in Eya protein in the dorsally shifted ol and a narrowing of the expression domain throughout the mdh and pc (Figure 5A; Rogge et al. 1995). In pnt mutants we see similar changes to Eya protein distribution in the ol, complete loss in the mdh and normal expression in the anterior-most portion of the head including the pc and pl (Figure 5B). While these loss-of-function effects are not identical, they are more similar than one might expect from two genes that are reportedly direct antagonists of one another. In a similar effort, Anderson et al conducted a screen in the embryo to identify transcriptional regulators of dac. Since neither aop/yan nor pnt were identified in that effort, we are confident that these mutants represent strong candidates for regulation of eya (Anderson et al. 2006). When we screened late stage embryos (post germband retraction) we saw very similar effects on eya expression presumably due to the breakdown in EGFR signaling. The developmental and patterning defects that we observe are ones that one would expect from loss of EGFR (Figure S2).

Figure 5.
EGFR regulation of eya in the embryo and eye. (A–D) Eya distribution in embryos that are either mutant for overexpressing activated transgenes of aop or pnt. Anterior is to the left. (E and H) Eya expression in eye discs overexpressing either ...

We used an antibody that recognizes the Aop/Yan protein to determine the aop/yan expression pattern in stage 9 embryos. We observe uniform expression across the head of embryos with particularly prominent expression within the mdh (Figure 5C). A pnt-lacZ stock was used to also determine the pnt expression pattern at this stage. pnt expression appears more restricted in the head, predominantly seen in two patches flanking the pc (Figure 5D).

Using an Ac5C-GAL4 driver we overexpressed aop and pnt throughout the developing embryo and assayed for changes in Eya protein distribution. When aop is expressed throughout the embryo we observe a broadening of the eya expression pattern throughout the visual primordium and mdh: an effect that is opposite to aop loss-of-function (Figure 5E). Interestingly, pnt overexpression results in an Eya protein distribution pattern that, aside from the striking increase in Eya at the anterior-most portion of the head, resembles the aop mutant (Figure 5F). This is consistent with an antagonistic relationship between these two genes. However, from this assay it appears that the relationship may be unidirectional since aop overexpression does not mimic pnt loss of function. This may be due to functional redundancy within the Ets family of transcription factors at sites which aop/yan regulates eya or a limitation in the ability of Yan protein itself to fully out-compete Pnt protein for regulatory binding sites.

Normally, aop is expressed at a high level posterior to the morphogenetic furrow where it serves to repress premature neuronal fate specification (Figure 6A; Rebay and Rubin 1995). Since we identified aop in our screen and since both Aop and Eya proteins have partially overlapping distribution patterns posterior to the morphogenetic furrow, we were interested in determining if EGF Receptor signaling through aop also regulates eya expression in the eye. We carried out ey-GAL4 and GMR-GAL4 driven overexpression experiments in the eye with activated aop and pnt responder lines. Expression of aop leads to a dramatic reduction in the size of the eye field (Figure 5, G and H). While Eya protein is still distributed within the anterior remnants of the eye field, the pattern is different than that seen in discs that express other genes such as so, osa or dve (compare 5G to 4D–F). Specifically, eya expression is not seen fully behind the small furrow suggesting that, in this example, the very small eye field may be due to a direct loss of eya. With surprising similarity, overexpression of a UAS-pnt transgene caused a dramatic reduction in the size of the adult eye (Figure 5K compare to 5H). However, unlike aop, Eya protein is still present ahead of and behind the distorted furrow within the reduced eye field (Figure 5J). Overexpression of either aop or pnt in developing photoreceptors via GMR-GAL4 resulted in animals with rough eyes (Figure 5, I and L). The rough eyes are likely to be due, in part, to alterations in Eya and So protein levels (Figure 6).

Figure 6.
aop/yan overexpression in the retina alters Eya, So and Dac distribution. (A–A”) Immunoflourescent image of wild-type eye imaginal disc stained with antibodies that recognize Yan and So proteins. Orange arrowhead denotes morphogenetic ...

Yan/Aop regulates the expression of so and eya but not dac:

Forced expression of aop in all developing photoreceptors via the GMR-GAL4 driver resulted in severe retinal defects. We assayed for changes in Eya protein distribution in response to activated aop overexpression and observed a marked, uniform depletion of Eya behind the furrow that began about 10 cell diameters behind the furrow (Figure 6B, yellow bracket). Since so, eya, and dac have a complex, position dependent regulatory relationship within the developing eye and since so and dac were also identified in our screen for regulators of eya we assayed for So and Dac protein distribution in aop over-expressing retinas. As with Eya we observed downregulation of So, however this effect was delayed in developmental time and occurred in much more posterior regions of the eye disc (Figure 6C yellow bracket). Dac, on the other hand showed no significant reduction within its normal expression domain. Instead, we observed rather patchy ectopic dac expression in the posterior-most region of the eye disc (Figure 6D, yellow asterisks). The upregulation of dac in response to the loss of so in the most posterior sections of the retina is consistent with our previous findings that normally So can function to repress dac in this region of the developing eye (Salzer and Kumar 2009).

Next we generated aop1 loss-of-function mosaic clones in the developing retina. In agreement with previous reports, removal of aop leads to a disorganization of the retina and an increase in photoreceptor cells (O'Neill et al. 1994). We extended these observations by demonstrating that Eya protein levels are elevated in aop1 clones behind the furrow (Figure 7, A–H). This finding suggests that in addition to the modification of Eya activity by MAPK phosphorylation (Hsiao et al. 2001), the EGFR pathway also regulates the expression levels of the eya transcriptional unit. In addition it appears that So protein levels are also elevated in aop clones (Figure 7, I–L) suggesting that either EGFR signaling via Aop regulates both so and eya or there is a serial link between these factors. It should be noted that we also observe an increase in ELAV protein within cells that are mutant for aop/yan. Since both eya and so are required for the maintenance of photoreceptor fate (Pignoni et al. 1997) it is possible that these factors regulate a number of neuron/photoreceptor specific genes including elav. As the down-regulation of aop/yan results in an increase in Eya and So protein levels it is possible that this in turn leads to an increase in ELAV protein levels. Downstream of both factors lies dac, which is regulated by the So-Eya composite transcription factor (Pappu et al. 2005). Interestingly, in aop clones, which have elevated levels of both so and eya, we do not see the expected elevation of Dac protein levels. Instead we observe a complete loss or severe downregulation of dac expression within aop clones (Figure 7, M–P arrowhead). As distinct regulatory relationships exist among the So-Eya-Dac subcircuit within different spatial regions of the eye field (Salzer and Kumar 2009) and since dac genetically interacts with the EGFR signaling cascade (Mardon et al. 1994), the loss of dac expression in aop clones is likely the result of the combined activities of the EGFR pathway and the So-Eya complex.

Figure 7.
Aop/Yan regulates the RD genes eya, so, and dac. (A–P) Immunofluorescence images of aop1 mutant clones in the eye imaginal disc. (A–H) Clones are marked by the absence of GFP and stained with antibodies that recognize Eya and Elav proteins. ...

The removal of pnt in the eye should result in the loss of both eya and so expression. And that is indeed what we observe. In clones that are lacking pnt activity, the levels of both So and Eya proteins are reduced but not eliminated (Figure 8). This is consistent with reports in which the loss of pnt results in the reduction but not completely loss of photoreceptor development (O'Neill et al. 1994). This result suggests that the loss of photoreceptor neurons in pnt mutants may in fact be due to the loss of eya and so expression.

Figure 8.
So and Eya protein levels are reduced in pnt clones. (A–D) Immunofluorescence images of pnt1 mutant clones in the eye imaginal disc. Clones are marked by the absence of GFP and stained with antibodies that recognize So and Eya proteins. Arrows ...

DISCUSSION

In this report we describe 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. We identified putative regulators by the loss or expansion of Eya protein distribution within the embryonic head of stage 9 loss-of-function mutants. Our 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 (Chen et al. 1999; Curtiss and Mlodzik 2000; Hsaio et al. 2001; Pappu et al. 2003; Firth and Baker 2009). We did not recover mutations in any of known Wingless pathway members. This was slightly unexpected as Wnt signaling and eya are known to reciprocally regulate each other (Hazelett et al. 1998). This result could imply, however, that eya is regulated differently in diverse tissues.

A screen similar to the one described here successfully identified the TGFβ pathway as an important upstream regulator of another retinal determination gene, dachshund (Anderson et al. 2006). 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 report; (Anderson et al. 2006). This is unsurprising as the expression pattern of eya, when compared to dac, is considerably more dynamic, at least within the embryonic head.

We were particularly interested in 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 (Figure 9; Hsiao et al. 2001). 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 (Hsiao et al. 2001; Silver et al. 2003).

Figure 9.
Model for EGFR mediated regulation of RD cofactors so and eya. Note that EGFR regulates Eya activity via phosphorylation by MAPK and also through transcriptional regulation at the genetic level. It remains unclear if the transcriptional regulation of ...

Our findings suggest that the EGFR pathway is also required to regulate eya transcription (Figure 9). This is consistent with findings that eya expression is lost in mago clones, which reduce Ras signaling (Firth and Baker 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 (Pignoni et al. 1997; Salzer and Kumar 2009). 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 (O'neill et al. 1994; Rebay and Rubin 1995). We see that, 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 (Figure 9).

Unexpectedly, we find 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. As our prior work has recently shown, the So-Eya-Dac subcircuit is under complex regulatory control (Salzer and Kumar 2009). The work presented here suggests that still greater complexity exists in the form of differential regulation by signal transduction cascades both at transcriptional and post-translational levels.

Acknowledgments

We thank Kavita Aurora, Michael O'Connor, Ilaria Rebay, and the Bloomington Stock Center for fly strains and reagents. We also thank Sally Salvador for technical help with the genetic screen and two anonymous reviewers for helpful suggestions and comments on this manuscript. The National Institutes of Health Genetics Training Grant awarded to Indiana University supports C.L.S. This work is also supported by a grant to J.P.K. from the National Eye Institute (R01 EY014863) and the Faculty Research Support Program at Indiana University.

Notes

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.110122/DC1.

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