So-VP16 partially restores eya expression and rescues eya² mutants

In third larval instar retinas eya is expressed in a small stripe of cells ahead of the advancing morphogenetic furrow, in differentiating photoreceptor, cone, and pigment cells, and in the developing ocelli (Fig 1A and 1B) [11]. In the eya² mutant eya expression is completely lost from the eye field (Fig 1C and 1D). We first set out to determine if the So consensus sites and regions of So ChIP peaks that are found outside of the original 322bp enhancer are functional. To do this we attempted to rescue the eya² mutant by forcibly expressing a So-VP16 chimeric construct in the developing eye with an ey-GAL4 driver. This protein is capable of fully restoring eye development to so¹ mutants [27] and activates a luciferase reporter at levels that are 20-fold higher than So alone and 5-fold higher than the So-Eya complex (Fig 1G). Based on these data we reasoned that So-VP16 serves as a strong transcriptional activator and therefore is a suitable substitute for the So-Eya complex (So-VP16 = So-Eya). Expression of So-VP16 partially restores both eya expression and eye development to 62% of the 57 animals that we examined (Fig 1E and 1F; S1A–S1C Fig). Consistent with being a very weak activator, expression of wild type So alone is insufficient to restore either eya expression or eye development to eya² mutants (Fig 1G; S1D and S1E Fig) [27]. These results led us to initially conclude that additional So-responsive enhancer element(s) are present within the eya locus.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. So-VP16 reactivates eya expression in the retina of eya² mutants.

(A,C,E) SEM images of adult female Drosophila compound eyes and heads. (A) wild type. (C) eya². (E) eya² mutants in which expression of UAS-So-VP16 partially restores eye development. (B,D,F) Light microscope images of third instar eye-antennal discs—eya expression is detected by antibody staining against Eya protein. (B) Wild type expression of eya in the compound eye and ocelli. (D) Loss of eya expression in the eye portion of disc in eya² mutants. Expression with the ocelli is maintained in the mutant. (F) Partial restoration of eya expression in the eye portion of disc upon expression of UAS-So-VP16. Anterior is to the right in adult head and imaginal disc images. At least 30 adult flies and developing imaginal discs were examined for each genotype with 57 adult eya²; ey-GAL4, UAS-So-VP16 flies being scored for rescue of eye structure (G) Luciferase assay quantifying activation strength of So-VP16. Y-axis is relative luciferase units (RLU). Three biological replicates were conducted for each experiment. Error bars in panel G represent standard deviation. Scale bar, 100μm

https://doi.org/10.1371/journal.pgen.1006462.g001

Newly identified regulatory elements are dynamically regulated during larval eye development

In order to identify regulatory elements that are responsive to the So-Eya complex we used the osm-6 gene and a CTSF insulator site to define the 5`and 3`boundaries respectively of the eya locus and then cloned fragments of DNA between these two genomic markers ahead of a minimal hsp70 promoter and a lacZ reporter (Fig 2A). These constructs were inserted into the same genomic coordinates (attP-3BVK00033—cytological position 65B2) using the PhiC31 integrase system to maintain similar expression levels across reporters. Wandering third instar eye-antennal imaginal discs were then examined for lacZ reporter expression. We identified six genomic fragments that are capable of driving expression of the reporter in portions of the endogenous eya pattern (Fig 2B–2G). Three of these fragments (PSE, 1, and E) have been previously identified as enhancers controlling eya expression in the retina [30,31,35]. The PSE, which stands for photoreceptor specific enhancer, drives expression solely in cells behind the morphogenetic furrow (Fig 2A and 2B) [35] while fragment 1 (also called IAM for immediately anterior to the morphogenetic furrow) drives expression ahead of the advancing morphogenetic furrow and in differentiating cells (Fig 2A and 2C) [35]. Fragment E (for extant) is the enhancer that is deleted in eya² mutants (Fig 2A and 2D) [30,31]. Our sequence analysis indicates that the fragment is 319bp in length (and not 322bp as originally reported). Fragments 2, 3 and 4 are three new retinal enhancers that control eya expression in the developing eye (Fig 2A and 2E–2G).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Multiple enhancers control expression of eya in the developing eye.

(A) Illustration of the genomic map of the eya locus—representation is not to scale. Sequences and genomic location of each fragment are provided in supplementary materials and methods S1-3. Size of each fragment (bp) is indicated within each bar. Blue bars = individual retinal enhancers. PSE = photoreceptor specific enhancer previously identified and named by Graeme Mardon’s group in [20]. E = 319bp extant enhancer previously identified in [11]. Enhancer 1 (IAM) = immediately ahead of morphogenetic furrow enhancer was previously identified and named by Graeme Mardon’s group in [20]. We refer to this enhancer as 1 as it shows a different expression pattern than previously reported. 2–4 represent newly identified enhancer elements. Orange bar = composite enhancer, purple bar = enhancer 1+E, grey bars indicate regions that do not drive expression in the retina including the fragment used as the 319bp spacer, asterisks = So binding sites, red bars = regions of So ChIP peaks. eya¹ and eya² deletions are indicated by red lines immediately ahead of exon 1 (B-G) Light microscope images of third instar eye-antennal discs. All images represent lacZ reporter expression in a wild type genetic background. LacZ reporter activation is indicated by antibody staining against β-galactosidase. White arrowheads mark the position of the morphogenetic furrow. (B) The PSE enhancer drives expression of the reporter only in cells that lie posterior to the morphogenetic furrow. (C) Enhancer 1 (also called IAM) drives expression in cells ahead and behind the morphogenetic furrow. (D) The 319bp extant enhancer drives weak reporter expression in cells ahead and posterior to the morphogenetic furrow. (E) Enhancer 2 drives expression of the reporter in cells anterior and posterior to furrow. (F) Enhancer 3 drives expression in cells ahead and behind the morphogenetic furrow. (G) Enhancer 4 drives expression only in cells posterior to the morphogenetic furrow. No single enhancer element fully recapitulates endogenous Eya expression. Anterior is to the right in imaginal disc images. At least 30 imaginal discs were examined for each genotype. Scale bar, 100μm

https://doi.org/10.1371/journal.pgen.1006462.g002

We next determined the temporal and spatial expression patterns of each individual fragment and compared these patterns to endogenous eya expression. Eya protein is present in the wild type eye disc as early as 48hrs AEL (early 2^nd instar, Fig 3A) and continues to be expressed broadly at 72hrs AEL (early 3^rd instar, Fig 3B). By the late third larval instar stage eya expression is restricted to a narrow band of cells ahead of the morphogenetic furrow and to all differentiating photoreceptor and cone cells (Fig 3C). No single individual fragment fully recapitulates the endogenous eya expression pattern. For example, reporter expression driven by fragment 1 is temporally and spatially delayed compared to wild type eya expression meaning that although it is activated in a few eya expressing cells early in development, it is not until late third instar that expression starts to coincide with the spatial pattern of endogenous eya (Fig 3D–3F, Table 1). In contrast, while reporter expression driven by fragment E coincides with early endogenous eya, its late expression is weak in intensity and appears mottled (Fig 3G–3I, Table 1). Lastly, the bulk of fragment 2 driven expression within younger discs is in eya negative cells while in later discs reporter expression does coincide with the endogenous eya gene (Fig 3J–3L, Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. The composite enhancer controls all eya expression within the developing eye.

(A-U) Light microscope images of developing eye-antennal discs. Images of imaginal discs at 48hrs and 72hrs AEL were taken at 20X while images of wandering third instar larvae were taken at 10X. AEL = after egg laying. Red = Eya protein, green = β-galactosidase, yellow = positions of co-localization between Eya and β-galactosidase. Arrowhead marks the position of the morphogenetic furrow. All enhancer-lacZ reporters are placed in a wild type genetic background. (A-C) Localization of Eya protein in developing wild type retinas at different developmental time points. (D-F) Enhancer 1 dependent expression is activated in a few Eya expressing cells early in development and co-localizes with Eya posterior to the morphogenetic furrow late in development. (G-I) Extant enhancer dependent expression co-localizes with Eya and is robust early in development but becomes weaker and sparse as development proceeds. (J-L) Enhancer 2-dependent expression is largely present in non-eya expressing cells early in development. Co-localization with Eya can be seen in cells anterior and posterior to the furrow later in development but a significant portion of reporter expression still present in non-eya expressing cells. (M-O) Composite enhancer-dependent expression shows co-localization with Eya protein throughout all stages of larval eye development. This is the only construct to fully recapitulate temporal and spatial eya expression. (P-R) Enhancer 3-dependent expression is largely present in non-eya expressing cells throughout development. Some co-localization with Eya protein is seen at later stages in cells anterior and posterior to the furrow. (S-U) Enhancer 4-dependent expression co-localizes with a few Eya expressing cells posterior to the furrow late in development. Anterior is to the right in imaginal disc images. At least 30 imaginal discs were examined for each genotype and developmental time point. Scale bar, 50μm

https://doi.org/10.1371/journal.pgen.1006462.g003

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Enhancer Expression and Rescue.

For each rescue experiment two-three female eyes were photographed with a scanning electron micrograph. We manually counted the number of ommatidia for each eye and calculated both averages and standard deviations (listed within table). The raw ommatidia counts for the rescue experiments are as follows: eya¹; enhancer 1—eya RB cDNA (141, 235, 190), eya²; enhancer 1—eya RB cDNA (322, 245), eya¹; enhancer E—eya RB cDNA (39, 38, 35), eya²; enhancer E—eya RB cDNA (358, 372, 362), eya¹; enhancer 1+E+2—eya RB cDNA (792, 730, 762), eya²; enhancer 1+E+2—eya RB cDNA (757, 825, 829), eya¹; enhancer 1+E—eya RB cDNA (376, 324, 298), eya²; enhancer 1+E—eya RB cDNA (635, 584, 636), eya¹; enhancer 1+spacer+2—eya RB cDNA (433, 433, 432), eya²; enhancer 1+spacer+2—eya RB cDNA (443, 448, 556).

https://doi.org/10.1371/journal.pgen.1006462.t001

Since each of these three fragments (1,E,2) does mimic a specific temporal and/or spatial aspect of eya expression we hypothesized that these enhancers, which lie adjacent to each other, might function cooperatively to control all temporal and spatial aspects of eya expression. To test this model we generated a single 1181bp fragment consisting of fragments 1, E, and 2 and as predicted this composite enhancer fully recapitulates the temporal and spatial expression pattern of eya within the developing eye (Fig 3M–3O, Table 1). To rule out position dependent effects we inserted this construct into a second genomic landing site (attP-9A VK00019—cytological position 68D2) and observe that the expression pattern of this insertion is identical to the original insertion and recapitulates endogenous eya expression (S2 Fig). It appears that the temporal expression of the composite enhancer is the sum or addition of the individual elements. And interestingly, recreating the genomic organization of these three cis-regulatory elements eliminates the ectopic expression from the eye-antennal disc (Fig 3J–3O, Table 1). Since the composite enhancer recapitulates the entire eya expression pattern it is possible that fragments 3, 4, and PSE are functionally redundant. Consistent with this model, the expression patterns controlled by these fragments are fully covered by the composite enhancer (Fig 3P–3U, Table 1).

The composite eya enhancer can fully rescue eye development in eya¹ and eya² mutants

We then set out to test if the composite enhancer is sufficient to fully rescue the no-eye phenotypes of eya² and eya¹ mutants. The original characterization of the eya¹ mutant indicated two chromosomal aberrations are associated with this mutation. First, a chromosomal re-arrangement completely inverts the orientation of the eya locus within the left arm of the second chromosome. This is not thought to interfere with normal eya expression. Second, an approximately 1.5kb deletion was detected at the 5`end of the gene. The 319bp deletion in eya<sup>2</sup> lies within the larger ~1.5kb deletion in eya<sup>1</sup>. Thus the no-eye phenotype of eya<sup>1</sup> and eya<sup>2</sup> is thought to result from the disruption of the same regulatory sites [11,31]. To precisely determine the breakpoints of the eya<sup>1</sup> deletion in relation to the composite enhancer we isolated and re-sequenced the region around the transcriptional start site and determined that the deletion is actually 1826bp in length with the deletion extending 581bp upstream of the eya<sup>2</sup> deletion and 344bp downstream of the transcriptional start site. This deletion completely deletes the composite enhancer, the transcriptional start site, and a large portion of the eya RB transcript 5`UTR (Fig 2A). Using qRT-PCR we confirmed that the RB transcript is completely eliminated in eya¹ mutants and drastically reduced in eya² mutants (Fig 4A). The RA transcript is also greatly reduced, but not eliminated, in both mutant alleles suggesting that the composite enhancer regulates both eya promoters (Figs 2A and 4A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. The composite enhancer fully restores eye development to eya¹ and eya² mutants.

(A) qRT-PCR quantification of eya RA and RB transcript levels in eye-antennal discs. Y-axis measures the relative expression levels of each transcript. Raw data from single runs from three biological replicates were used to generate the graph. Error bars represent standard error. (B-O) SEM images of adult Drosophila compound eyes and heads from enhancer cDNA fusion rescue experiments. Each enhancer is driving expression of the eya RB isoform within the developing eye of eya¹ and eya² mutants. (B,I) Enhancer 1—eya RB cDNA fusion partially rescues 100% of animals examined. (C,J) Enhancer E—eya RB cDNA fusion partially rescues 100% of animals examined. Rescue efficiency is significantly reduced in eya¹ background. (D,K) Enhancer 2—eya RB cDNA fusion does not rescue either eya¹ or eya² mutants. (E,L) Composite enhancer—eya cDNA fusion fully rescues 100% of animals examined to wild type eye size. (F,M) Enhancer 1+E—eya cDNA fusion partially rescues 100% of animals examined. (G,N) Enhancer 3—eya cDNA fusion does not rescue eya¹ or eya² mutants. (H,O) Enhancer 4—eya cDNA fusion does not rescue eya¹ or eya² mutants. Anterior is to the right in all adult head images. At least 100 adult flies were examined qualitatively for each genotype. Quantification of rescue (assayed by number of ommatidia) of a subset of adults is provided in Table 1. Scale bar, 100μm.

https://doi.org/10.1371/journal.pgen.1006462.g004

To test whether fragments 1, E, and 2 are sufficient to rescue the two eya mutants, each enhancer element, as well as the full composite enhancer, was cloned upstream of a minimal hsp70 promoter and the eya RB cDNA. Using the PhiC31 integrase system these constructs were inserted into the same genomic location that we used for the original lacZ reporter expression analysis (attP-3BVK00033—cytological location 65B2). For all rescue experiments at least 100 adult flies were initially assayed qualitatively for the restoration of eye development. For the rescue quantification in Table 1 the number of ommatidia in adult right eyes from 2–3 individual female flies were counted and compared to wild type. The number of ommatidia per rescue is presented as an average of the 2–3 individuals. A wild type eye from a female fly is defined as having between 750 and 800 ommatidia [36].

Both fragments 1 and E are capable of partially restoring eye development in 100% of eya² and eya¹ mutants. Fragment 1 restores eye size to approximately 38% of wild type in eya² and 25% in eya¹ (Fig 4B and 4I, Table 1). Enhancer E restores eye size to approximately 49% of wild type in eya² but less than 1% in eya¹ (Fig 4C and 4J, Table 1). Expression from fragment 2, on its own, fails to rescue either mutant (Fig 4D and 4K, Table 1). Consistent with our expression analysis, the full composite enhancer fully restores eye development to 100% of both eya mutants (Fig 4E and 4L, Table 1). And finally, neither fragment 3 nor 4 are capable of rescuing the no-eye phenotype of either mutant (Fig 4G, 4H, 4N and 4O, Table 1). The majority of fragment 3 driven expression is outside of the endogenous eya expression pattern and would therefore not be predicted to restore eye development to eya mutants (Fig 3P–3R, Table 1). The inability of fragment 4 to rescue eye development stems from the fact that it is normally expressed only in differentiating cells posterior to the morphogenetic furrow (Fig 3S–3U, Table 1). Neither the furrow nor differentiated photoreceptor cells are present in either eya¹ or eya² mutants [11].

The lack of any discernable rescue by fragment 2 and its inappropriate expression pattern initially indicated that it may not function as an enhancer. Instead, its proximity to the transcriptional start site of eya RB, suggested that it might serve as a basal core promoter. To test this idea we placed fragment 2 and the composite enhancer into a plasmid that contains a lacZ reporter but lacks a minimal promoter. Under these conditions fragment 2 is still capable of driving lacZ expression in the developing eye but only in developing photoreceptors (S3A and S3B Fig). In contrast, lacZ reporter expression driven by the composite enhancer is identical to the construct that contained the minimal hsp70 promoter fragment (S3C and S3D Fig). These data support the proposal that fragment 2 functions, in part, as a basal promoter. As such we then tested the model that all pertinent regulatory information may reside only in fragments 1 and E. We first examined lacZ reporter expression with a fragment that contained segments 1 and E only and as expected this construct fully recapitulates endogenous eya expression (S3E and S3F Fig). We next attempted to rescue both eya¹ and eya² mutants with this shorter fragment. While we observed rescue in 100% of animals it only restores eye size to 82% of wild type in eya² and 44% in eya¹ mutants (Fig 4E, 4F, 4L and 4M, Table 1). This is unlike the composite enhancer, which completely restores eye size to the both eya mutants. This suggests that, in addition to functioning as a basal core promoter, fragment 2 does indeed contain regulatory information that is necessary for robust eya expression.

We were intrigued by the differences in rescue efficiency of our constructs in eya¹ and eya² mutants. Since the endogenous transcriptional start site for the RB transcript is intact in the eya² mutant but is deleted in the eya¹ mutant we hypothesized that the higher degree of rescue in the eya² mutant is due to a reactivation of the endogenous eya gene. Using qRT-PCR we measured eya RB transcript levels within eya mutants that have been rescued by expression from enhancer E. This enhancer was chosen since it showed the most dramatic difference in rescue efficiency. As predicted, we observe that expression of the eya RB cDNA initiates a positive feedback loop on the endogenous locus and reactivates eya expression in eya² but not eya¹ (Fig 4A, 4C and 4J). In the eya² mutant, fragments 1 (IAM), 2, 3, 4, and PSE are present and one or more of these could be targets of the auto-regulatory loop. To test this possibility we brought combinations of rescue constructs together within a single eya¹ animal and asked if the degree of rescue could mimic that of the extant enhancer rescue of eya². We combined the extant enhancer (E, Fig 5A) with each of the other enhancer elements (Fig 5B–5F) and observed a synergistic increase in the quality of rescue only with enhancer 1 (Fig 5G). The quality of eye restoration did not improve by combining the other elements with the extant enhancer (Fig 5H–5K). These data, when combined with the reporter expression and rescue results, suggest that enhancer 1 mediates the Eya-dependent auto-regulatory loop.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Cooperative interactions between enhancers 1 and E drive eye development.

(A-K) Light microscope images of adult Drosophila compound eyes and heads from single and combination enhancer—eya RB cDNA fusion rescue experiments. Each enhancer is driving expression of the eya RB isoform within the developing eye of eya¹ mutants. (A) eya¹/eya¹; extant enhancer E—eya RB cDNA. Expression of the eya RB cDNA driven by the extant enhancer alone weakly rescues 100% of animals examined. (B) eya¹/eya¹; enhancer 1—eya RB cDNA. Expression of the eya RB cDNA driven by enhancer 1 alone partially rescues 100% of animals examined. (C) eya¹/eya¹; enhancer 2—eya RB cDNA. Expression of the eya RB cDNA driven by enhancer 2 alone does not rescue the no-eye phenotype. (D) eya¹/eya¹; enhancer 3—eya RB cDNA. Expression of the eya RB cDNA driven by enhancer 3 alone does not rescue the no-eye phenotype. (E) eya¹/eya¹; enhancer 4—eya RB cDNA. Expression of the eya cDNA driven by enhancer 4 alone does not rescue the no-eye phenotype. (F) eya¹/eya¹; enhancer PSE—eya RB cDNA. Expression of the eya RB cDNA driven by the PSE enhancer alone does not rescue the no-eye phenotype. (G) eya¹/eya¹; enhancer 1—eya RB cDNA/extant enhancer E—eya RB cDNA. Combining enhancer 1—eya RB cDNA and enhancer E—eya RB cDNA constructs increases the quality of rescue as demonstrated by the larger eye size in 100% of animals examined. (H) eya¹/eya¹; enhancer 2—eya RB cDNA/extant enhancer E—eya RB cDNA. Combining enhancer 2—eya RB cDNA and enhancer E—eya RB cDNA constructs does not increase the quality of rescue over the extant enhancer alone. (I) eya¹/eya¹; enhancer 3—eya RB cDNA/extant enhancer E—eya RB cDNA. Combining enhancer 3—eya RB cDNA and enhancer E—eya RB cDNA constructs does not increase the quality of rescue over the extant enhancer alone. (J) eya¹/eya¹; enhancer 4—eya RB cDNA/extant enhancer E—eya RB cDNA. Combining enhancer 4—eya RB cDNA and enhancer E—eya RB cDNA constructs does not increase the quality of rescue over the extant enhancer alone. (K) eya¹/eya¹; enhancer PSE—eya RB cDNA/extant enhancer E—eya RB cDNA. Combining enhancer PSE—eya RB cDNA and enhancer E—eya RB cDNA constructs does not increase the quality of rescue over the extant enhancer alone. Anterior is to the right in adult head images. At least 100 adult flies were examined for each genotype. Scale bar, 100μm.

https://doi.org/10.1371/journal.pgen.1006462.g005

The no-eye phenotype of eya² mutants results from disrupting neighboring enhancers

The ability of enhancer 1 to partially restore eye development to eya¹ and eya² mutants was of particular interest to us since eye development is completely blocked in the eya² mutant despite the continued presence of enhancer 1. We hypothesized that the loss of eye development is due to the combined loss of enhancer E and a disruption of enhancer 1 activity. To test this model we recapitulated the genomic organization of the eya² mutant by fusing enhancers 1 and 2 together. When the enhancers are placed in this configuration expression of the lacZ reporter is lost throughout young eye discs and ahead of the furrow in third instar discs. Expression of the reporter only remains in some differentiating cells posterior to the furrow (Fig 6A–6C, Table 1). Consistent with the loss of expression in undifferentiated cells, this construct drives reporter expression in a very small number of cells in the eya² mutant (Fig 6D). The loss of expression in undifferentiated cells prevents this construct from rescuing the eya² mutant (Fig 6E, Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Spacing between enhancers is required for eya expression and function.

(A-D, F-I, K-N,Q,R) Light microscope images of developing eye-antennal discs. Images of imaginal discs at 48hrs and 72hrs AEL were taken at 20X while images of wandering third instar larvae were taken at 10X. Red = Eya, green = β-galactosidase, yellow = regions where Eya and β-galactosidase co-localize. White arrowheads mark the position of the morphogenetic furrow. Each enhancer is driving expression of lacZ within wild type eye-antennal discs. AEL = after egg laying. (E,J,O,S) SEM images of adult Drosophila compound eyes and heads from enhancer—eya RB cDNA rescue experiments. Each enhancer is driving expression of the eya RB isoform within the developing eye of either eya¹ or eya² mutants. (P) SEM image of adult Drosophila compound eyes and heads from enhancer 1+spacer+2—eya RB cDNA rescue experiments of eya¹ mutants. (A-C) Expression driven by enhancer 1+2 is only activated late in eye development in some eya expressing cells posterior to the furrow. (D) Expression driven by enhancer 1+2 is activated in very few cells in eya² mutant discs. (E) The enhancer 1+2—eya RB cDNA does not rescue eya² mutants. (F-H) Expression driven by enhancer 1+5bp+2 is activated mostly in non-eya expressing cells early in development while later activation is seen in eya expressing cells both anterior and posterior to the furrow. (I) Expression driven by enhancer 1+5bp+2 is activated weakly throughout the eye disc of eya² mutants. (J) The enhancer 1+5bp+2—eya RB cDNA does not rescue eya² mutants. (K-M) Expression driven by enhancer 1+spacer+2 restores some early expression in eya expressing cells but does not fully recapitulate eya expression at all stages of development. (N) Expression driven by enhancer 1+spacer+2 is strongly activated throughout the eye disc of eya² mutants. (O) Enhancer 1+spacer+2—eya RB cDNA partially rescues 100% of eya² mutants suggesting a restoration of function. (P) Enhancer 1+spacer+2—eya RB cDNA partially rescues 100% of eya¹ mutants suggesting a restoration of function. (Q-R) The neutral 319bp of DNA that was used to construct 1+spacer+2 does not drive reporter activation on its own in either wild type or eya² discs. (S) The 319bp spacer does not rescue eya² mutants. Anterior is to the right in adult head and imaginal disc images. At least 100 adult flies and 30 imaginal discs were qualitatively examined for each genotype and at each developmental time point. Panel A-D, F-I, K-N, Q-R Scale bar, 50μm. Panel E,J, O,P,S Scale bar, 100μm

https://doi.org/10.1371/journal.pgen.1006462.g006

The inability of this construct to properly drive lacZ and eya cDNA expression could be due to either the unintended creation of a synthetic binding site for a transcriptional repressor at the junction where enhancers 1 and 2 meet or there might be a need for some amount of genomic space between the two enhancers. To test the first possibility we placed a BamHI restriction site between enhancers #1 and #2. Addition of this 5bp spacer restores expression to some cells in wild type discs and to a few cells in eya² mutant discs (Fig 6F–6I, Table 1). The expression pattern in wild type discs resembles that of enhancer 2 suggesting that insertion of the 5bps failed to allow for the early activation of enhancer 1. Consistent with this construct behaving similar to enhancer 2 we did not see any rescue of the eya² mutant (Fig 6J, Table 1). Since this construct failed to restore eya expression and eye development, we can rule out the possibility that a synthetically created repressor site is the underlying reason for the loss of eya expression in eya² mutants.

To test the latter hypothesis that a certain amount of genomic space is required between enhancers 1 and 2 we inserted a 319bp fragment of DNA (the size of enhancer E) between the two fragments in an effort to reinstate normal spacing. On its own this neutral sequence, which comes from intron 1 of the eya locus, does not direct expression of lacZ or rescue the eya² mutant (Figs 2A and 6Q–6S, Table 1). At 48hrs and 72hrs AEL the majority, but not all, of the reporter expression of 1+spacer+2 was still seen in non-eya expressing cells (Fig 6K and 6L, Table 1). However, by the late third larval instar reporter expression is now seen in the majority of Eya positive cells (Fig 6M, Table 1). Overall early reporter expression of 1+spacer+2 is similar to that of enhancer 2 alone while late reporter expression is comparable to enhancer 1 alone (compare to Fig 3D–3F and 3J–3L, Table 1). This construct can drive expression in and partially rescue both eya¹ and eya² mutants demonstrating that the reconstitution of spacing was sufficient to restore limited function to enhancers 1 and 2 (Fig 6O and 6P, Table 1). The restoration of eye size in eya² and eya¹ is approximately 64% and 58% of wild type respectively, compared to 100% for the composite (1+E+2) enhancer, suggesting that in addition to providing critical space between enhancers #1 and #2, enhancer E must also contain regulatory information necessary for robust eya expression (Table 1).

Activation of the composite enhancer is not So dependent

Since we are able to partially restore eya expression and eye development to eya² mutants through expression of the So-VP16 chimeric protein (Fig 1F and 1G) we reasoned that one or more of the newly discovered enhancers might be regulated by So. Three enhancers contain canonical So binding sites and So ChIP peaks are present within two other enhancers (Fig 2A) [33,34]. We first tested whether the So-VP16 chimeric protein is capable of activating the composite (1+E+2) enhancer. When forcibly expressed in the antennal disc under the control of the dpp-GAL4 driver, So-VP16 is surprisingly unable to activate the composite enhancer (S4A–S4D Fig, rose arrow). In contrast, forced Ey does activate the reporter suggesting that Ey, but not So, regulates the eya locus during eye development (S4E–S4H Fig, yellow arrow). To further test whether activation of any of the enhancers is So dependent we placed each of the lacZ reporter constructs into the so¹ mutant background and assayed for lacZ expression. so¹ mutants are viable, lack compound eyes, have a small eye disc, and have drastically reduced levels of so expression (S5 Fig) [10,37]. Any So dependent element should remain silent in this mutant background. However, all of the elements with the exception of enhancer 4 remain activated in so¹ mutant eye discs (Fig 7A–7F). Enhancer 4 drives expression exclusively in differentiating cells thus the lack of activation from this enhancer is most likely due to the fact that so¹ mutants lack photoreceptor, cone, and pigment cells. It is striking that the composite enhancer remains strongly activated in so¹ mutants (Fig 7D). To ensure that this is not due to residual So protein we examined lacZ expression driven by the composite enhancer in so³ null mutant clones. We again find that the composite enhancer is strongly activated in clones both ahead and behind the morphogenetic furrow (Fig 7G–7J). Thus, despite the presence of a So binding site and the apparent binding of So, the composite enhancer (which contains all regulatory information for proper eya expression) is not activated by So.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. eya retinal enhancers remain transcriptionally active in so mutants.

(A-F) Light microscope images of wandering third instar so¹ mutant eye-antennal discs. Each enhancer is driving expression of lacZ within so¹ eye-antennal discs. LacZ reporter activation is detected with an antibody that recognizes the β-galactosidase enzyme. (A) so¹; enhancer 1—lacZ. Reporter expression driven by enhancer 1 is activated only in cells at the far posterior edge of the eye disc. (B) so¹; enhancer E–lacZ. Reporter expression driven by the extant enhancer E is weakly activated throughout the eye disc. (C) so¹; enhancer 2 –lacZ. Reporter expression driven by enhancer 2 is strongly activated throughout the remaining eye disc. (D) so¹; enhancer 1+E+2 –lacZ. Reporter expression driven by the composite enhancer is strongly activated throughout the eye disc. (E) so¹; enhancer 3 –lacZ. Reporter expression driven by enhancer 3 is activated broadly throughout the entire eye-antennal disc. (F) so¹; enhancer 4 –lacZ. Reporter expression driven by enhancer 4 is not activated in the eye disc. (G-J) Light microscope images of wandering third instar eye antennal discs in which so³ null clones have been generated. The absence of GFP marks the position of clones lacking so. Green = GFP, red = Eya protein, blue = lacZ. Yellow arrows mark the position of so³ mutant clones in which Eya protein is present and the composite enhancer lacZ reporter is activated. White arrowheads mark the position of the morphogenetic furrow. Anterior is to the right in all imaginal disc images. At least 30 discs were examined for each genotype. Scale bar, 100μm.

https://doi.org/10.1371/journal.pgen.1006462.g007

So is not required for the initiation of eya expression but may be required for its maintenance

When we examined the potential activation of the composite enhancer in so³ null clones, we were quite surprised to see clones that contain Eya protein (Fig 7H). This clearly suggests that activation of reporters in so mutants is not due the persistence of lacZ protein although we cannot entirely rule that out. However, this result certainly was inconsistent with what we observed in late third instar whole mutant so¹ discs where Eya protein was completely missing in the eye portion of the disc. These data were also inconsistent with qRT-PCR data, which showed a dramatic reduction of eya transcript levels in so¹ mutants (S4 Fig). A possible explanation for these apparently contradictory observations could be that Eya protein expression is lost over the course of larval eye development. This could be the result of a requirement for So in the maintenance of eya expression, retinal progenitor cell death, a fate transformation, or a combination of all three [28,38]. Retinal progenitors have previously been defined as those proliferating in the most anterior regions of the eye disc and express Ey but lack So and Eya. Retinal precursors are defined as cells anterior to the morphogenetic furrow which express all three genes [39]. Support for the model that eya expression is lost over developmental time comes from three previously published observations: (1) eya expression is lost within the retinal field in roughly 50% of mid-late second larval instar so¹ eye-antennal discs [29]; (2) so¹ mutants undergo a significant wave of cell death that eliminates retinal progenitors in the growing eye field [10]; and (3) retinal progenitors within so and eya mutants undergo a fate transformation into head epidermis [28,38].

To test our hypothesis that loss of Eya in so¹ mutants is progressive we first re-examined eya expression in so¹ mutants over the course of larval eye development. Beginning at 72hrs AEL we found that 100% of so¹ mutant discs had strong Eya expression throughout the eye disc thereby demonstrating that So is not required for the initiation of eya expression (Fig 8A; S6 Fig). By 96hrs AEL eya expression weakens, is expressed in fewer discs, and is found in smaller and smaller populations of cells over time (Fig 8B–8D; S6 Fig). By 168hrs AEL the overwhelming majority of so¹ discs have completely lost eya expression within the retinal field (Fig 8E; S6 Fig). This analysis confirms that Eya protein is indeed lost over the course of larval eye development.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. Eya expression is lost progressively in so¹ mutants as a result of increased cell death in retinal progenitors.

(A-O) Light microscope images of developing so¹ mutant eye discs. (A-E) Green arrows indicate regions containing Eya protein. The amount of Eya protein is progressively lost in so mutant retinas. (F-O) Light microscope images of developing so¹ mutant eye discs showing positions of dying cells. Cell death (green) is marked by Dcp-1, Ey (magenta) marks the position of progenitor cells, and Eya (blue) marks the position of precursor cells. Cell death in so¹ mutants is elevated in both progenitor and precursor cells. (P-Q) Light microscope images of developing so¹, eya composite enhancer GAL4, UAS-p35. Cell death has been blocked in the eye disc of so¹ mutants by expression of the caspase inhibitor p35. Blocking death does not prevent the loss of eya expression in late third instar so¹ mutant discs. AEL = after egg laying. Anterior is to the right in all imaginal disc images. At least 30 discs were examined for each genotype and at each developmental time point. Scale bar, 50μm.

https://doi.org/10.1371/journal.pgen.1006462.g008

To determine whether the loss of Eya protein could be due to increased cell death in retinal progenitors, as suggested by previous studies, we conducted a temporal examination of cell death in so¹ mutants and find that retinal progenitors undergo significant cell death over the course of larval eye development (Fig 8F–8J). Using an antibody against Dcp-1, a marker of cell death, we observed increased cell death at 72hrs AEL in a large swathe of cells in the anterior most portions of the eye disc. At this point cell death seems restricted mostly to retinal progenitors outside the endogenous Eya expression domain as indicated by the expression of Ey but not Eya (Fig 8F and 8K). By 96hrs AEL the wave of cell death becomes broader and extends to the posterior margins of the disc to include both retinal progenitors and retinal precursors as indicated by the presence of both Ey and Eya expression (Fig 8G and 8L). It is important to note that 96hrs AEL is the first time point in which we see decreases in the expression of Eya protein. Finally, as development proceeds, the amount of cell death decreases and becomes restricted to the ventral most portions of the disc (Fig 8H–8J and 8M–8O). Although some Eya positive cells do appear to remain outside the population of dying cells it is clear that the majority of Eya expressing cells have been removed by these later time points consistent with the idea that retinal progenitors, and by default retinal precursors, have been cleared by cell death. These data also corroborate the qRT-PCR data from late so¹ mutant discs.

If the loss of Eya expression in so¹ mutants was solely the result of cell death of retinal progenitors then it follows that blocking cell death should restore eya expression to a subset of cells in late so¹ mutant discs as those cells would be saved earlier in larval eye development and then proceed to differentiate into retinal precursors and express eya. To test this hypothesis we blocked cell death by expressing P35, a well-known inhibitor of caspase dependent cell death, with an eya composite enhancer GAL4 driver. We saw no increase in eya expression at 120hrs AEL suggesting that the loss of eya in so¹ mutants is not simply the result of a clearing of retinal progenitors (Fig 8Q). However, we do see a significant number of cells still expressing ey indicating the continued presence of retinal progenitors that are not proceeding to differentiate into retinal precursors (Fig 8P).

We have previously shown that retinal progenitors within so and eya mutants undergo a cell fate transformation into head epidermis [28,38]. It is possible that after the wave of cell death the continued loss of eya expression in so¹ mutants may be the indirect result of this homeotic transformation. The non-ocular bristle and antennal selector gene cut (ct) and the head capsule selector gene orthodenticle (otd) have previously been shown to be de-repressed in eye to head epidermis transformations in eya² mutants [28]. We therefore examined expression of both genes in so¹ mutants. Concomitant with the decrease in eya expression, we saw a de-repression and expansion of both ct and otd throughout the entire eye disc (Fig 9A–9L). The de-repression of both genes initiates at 72hrs AEL in just a few cells of the eye disc but is more pronounced by 96hrs AEL (Fig 9D, 9E, 9G, 9H, 9J and 9K). Most striking is that starting at 120hrs AEL, when we first begin to see discs without any eya expression in the retinal field, ct and otd expression have expanded to cover the entire eye field (Fig 9F, 9I and 9L). Based on the continued presence of ey expression within the same portion of the eye disc it appears that the surviving retinal progenitor cells have undergone a transformation to head epidermis (Fig 8H). Furthermore, when we block cell death in the mutants, ct and otd are still expressed in the majority of cells within the disc again supporting the idea that these cells have undergone a cell fate transformation (Fig 9M). We believe it is this ongoing cell fate transformation that is blocking the continued expression of eya resulting in a loss of Eya protein in late stage so¹ mutant discs. However, additional studies are needed to fully determine if, and possibly to what extent, So might be required for the maintenance of eya expression later in larval eye development.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 9. Retinal progenitors within so¹ mutants progressively transform into head epidermis.

(A-M) Light microscope images of developing wild type, so¹, and so¹, eya composite enhancer GAL4, UAS-p35 eye-antennal discs. Green = Otd and red = Cut. Expression of both otd and cut is de-repressed within the eye field of so¹ mutant discs over the course of larval eye development. Although cell death has been blocked in the eye disc of so¹ mutants by the caspase inhibitor p35, expression of otd and cut is still de-repressed in retinal progenitors. AEL = after egg laying. Anterior is to the right in all disc images. At least 30 discs were examined for each genotype at each developmental time point. Scale bar, 50μm.

https://doi.org/10.1371/journal.pgen.1006462.g009

Finally, to ensure that the Eya protein we observed in so¹ mutants is not the result of residual levels of So protein activity we examined eya expression in so³ null mutant clones. Consistent with the analysis of so¹ discs, we found multiple so³ null clones in which Eya protein was still present (Fig 10A–10H, yellow arrows). We did, however, observe that the majority of large clones spanning the middle of the eye field contained no Eya protein (Fig 10A–10H, green arrows). The adult retinas of these animals often contain large patches of head epidermis protruding through the middle of the eye field (Fig 10I–10L, green arrows). We predict that these patches of head cuticle correspond to the clones in the disc that lack eya expression and thus are the result of a cell fate transformation.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 10. Eya protein is detected in non-transformed so³ mutant clones.

(A-L) Eye discs and adult heads of the genotype: eyflp; FRT42D so³ /FRT42D Ubi-GFP that contain so³ mutant clones. (A-H) Light microscope images of developing eye-antennal discs. Yellow arrows identify so³ clones that have not transformed into head epidermis and still express eya. Green arrows demarcate large so³ clones that are transforming into head epidermis and lack Eya protein. Red = F-actin, green = GFP, blue = Eya. (I-L) SEM images of adult Drosophila compound eyes and heads. Green arrows indicate regions of the compound eye that has transformed into head epidermis. Green arrows in panels I-L indicated regions of head epidermis bifurcating the retinal field. Anterior is to the right in all adult head and imaginal disc images. At least 30 adult eyes and imaginal discs containing so³ clones were examined. Scale bar, 100μm.

https://doi.org/10.1371/journal.pgen.1006462.g010

Together our lacZ reporter and cDNA rescue analyses suggest that a single 1181bp genomic fragment composed of three cis-regulatory elements is capable of controlling all eya expression in the developing retina. Furthermore, our combinatorial rescue analysis in eya¹ mutants suggests that although enhancer elements 1 and E sit adjacent to each other within the eya locus, these two elements are functioning as independent cis-regulatory elements. Additionally, we find that spacing between elements within the composite enhancer is critical for proper function. When fragments 1 and 2 are located adjacent to each other as is the case in eya² mutant animals these enhancers can no longer function to provide eya expression early in larval development leading to an adult no-eye phenotype. Finally, we find that loss of eya expression in so loss-of-function mutants is progressive and likely the result of increased cell death and a cell fate transformation. Although our data cannot rule out the possibility that So is required for the maintenance of eya expression during larval eye development it is clearly not required for its initiation. Given our identification of multiple independently functioning cis-regulatory elements within the eya locus and the potential differential requirement for So in its activation at later stages of eye development, eya regulation over the course of eye development is likely to be dynamic and require the input of different combinations of RD members and signaling pathways at different times and in different cell types for overall proper temporal and spatial expression.

Fly Strains

The following fly strains were used in this study: (1) eya¹, (2) eya², (3) so¹, (4) FRT42D so³/CyO, (5) FRT42D Ubi-GFP/CyO, (6) y w eyflp, (7) w¹¹¹⁸, (8) w;; ey-GAL4, (9) UAS-so^VP16, (10) y¹ M(vas-int.Dm))[2]ZH-2A w^*; PBac(y⁺-attP-3B)VK00033—BL24871, (11) y¹ M(vas-int.Dm)ZH-2A w^*; PBac(y⁺-attP-9A)VK00019—BL24866, (12) so¹, UAS-P35, (13) w¹¹¹⁸; eya composite enhancer-GAL4. Loss-of-function clones were generated with the following genotype: y w eyflp; FRT42D so³/FRT42D Ubi-GFP. All crosses were conducted at 25°C. BL = Bloomington Drosophila Stock Center

Antibodies, Dissections, and Microscopy

The following antibodies were used: (1) mouse anti-Eya (1:5, DSHB), (2) mouse anti-β-galactosidase (1:250, Promega), (3) chicken anti-β-galactosidase (1:800, Promega), (4) rat anti-Elav (1:100, DSHB). (5) rabbit anti-Dcp-1 (1:100, Cell Signaling Technologies). DSHB = Developmental Studies Hybridoma Bank. Fluorophore-conjugated secondary antibodies and phalloidin-fluorophore conjugates were obtained from Jackson Immuno Research Laboratories and Life Technologies. Imaginal discs were prepared as described previously in [47]. For dissections performed at specific time intervals, adult flies were placed in egg laying chambers and allowed to lay for 30–60 minutes on agar plates. Individual embryos were then transferred to individual microcentrifuge tubes with approximately 200ul of standard fly media. The tubes were then placed at 25°C and aged for the appropriate amount of time. Eye-antennal discs were photographed on a Zeiss Axioplan II compound microscope. For scanning electron microscopy, adult flies were serially incubated in 25% ethanol, 50% ethanol, 75% ethanol, 100% ethanol, 50% ethanol: 50% hexamethyldisilazane (HMDS), and then 100% HMDS, coated with gold-palladium, and viewed with a JEOL 5800LV SEM. For light microscopy of adult heads, flies were photographed on a Zeiss Discovery Microscope.

Luciferase Reporter Activation Assays

3mL of Drosophila Kc167 cells (approximately 1X10⁷ cells/mL) were transfected with a total of 400ng of plasmid DNA using the Qiagen Effectene Transfection Reagent (Cat. No. 301427). For each transfection, mt-GAL4 (136ng) was transfected along with the indicated UAS responder plasmids (64ng each) and ARE-luciferase (132ng). UAS-renilla (0.26ng) was also included in the transfection mix as a control for transfection efficiency. The plasmids were diluted in 98μL of Buffer EC, then 3.2μL of the Enhancer Solution was added to the dilution. The solution was incubated at room temperature for 5min. 10μL of Effectene Transfection Reagent was added to the dilution and the solution was incubated at room temperature for an additional 10min. The transfection solution was mixed with 600μL of Hyclone SFX Insect Culture Media (Cat. No. SH30278.02) and added drop-wise to the plated cells.

Following transfection the cells were incubated at 25°C for 20hr. Protein production was then induced by the addition of 1mM CuSO₄. Following induction cells were incubated at 25°C for an additional 24hr before harvesting for determination of luciferase activity. The luciferase activity was assayed using the Promega Dual Luciferase Reporter Assay System (Cat. No. E1910) and a Promega GloMAX 20/20 Luminometer (Model No. E5311). Cells were collected by centrifugation at 500g for 2min. The supernatant was removed and the pellet was re-suspended in 500μL of Passive Lysis Buffer (PLB) at the working concentration. The cells were lysed in the PLB for 20min at room temperature.

20μL of cell lysis solution was added to 100μL of Luciferase Assay Reagent II and mixed by pipetting for 10sec. The light output of the solution was measured in the luminometer once a second for 10s and the average output over the time period was recorded. This was the activity of the luciferase enzyme—the experimental result. 100μL of Stop and Glo Reagent was then added to the tube and mixed briefly by vortexing. The light output of the solution was once again measured, and the results were recorded as the output from Renilla enzyme—the transfection efficiency control. These two measurements were performed for each of three separate plates of independently transfected cells for each plasmid combination. The Relative Luciferase Units (RLUs) for each combination of plasmids were calculated by dividing the experimental light output (Luciferase) by the transfection efficiency control (Renilla) for each of the three independent transfections. Error bars in Fig 1 represent standard deviation

The target sequence for So and So-VP16 consists of five copies of the ARE element (GGT GTC AGG TTG CTC GAG) that is reported in [23,48] placed upstream of the luciferase gene within the pGL3 vector (Promega, catalog #E1751).

LacZ transcriptional reporters

For lacZ reporter analysis individual genomic fragments illustrated in Fig 2 were amplified from w¹¹¹⁸ genomic DNA and cloned into either p-lacZ.attB or pg-lacZ.attB plasmids (Konrad Basler, University of Zurich, Switzerland). Genomic fragment sequences are provided in S1 Table. Cloning strategies and primer sequences are listed in S2 Table. RED refers to standard restriction enzyme digestion and ligation into a multiple cloning site. Gateway refers to the Life Technologies Gateway Recombination Cloning system.

eya RB cDNA rescue plasmid

For the cDNA enhancer fusion rescue assay, a pg-eya RB+3’UTR cDNA.attB plasmid was created by modifying an existing pg-RFP.attB plasmid (derived from pg-lacZ.attB). The eya RB+3’UTR cDNA was first amplified by PCR from an existing pUAS-eya RB+3’UTR plasmid as an EcoRI-NdeI fragment and cloned into a pg-RFP.attB plasmid. Portions of the Gateway cloning cassette and hsp70 minimal promoter were then amplified from pg-RFP.attB as an EcoRI fragment and cloned ahead of the eyaRB+3’UTR cDNA. Primer sequences are listed in S2 Table.

Enhancer eya cDNA fusion rescue constructs

Putative enhancers (Fig 2A) were amplified from the appropriate p.lacZ.attB plasmid and cloned into the new pg-eya RB+3’UTR cDNA.attB plasmid using Gateway recombination cloning (Life Technologies). Gateway 5’ att primer sequence: 5`-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC AAC-3`and Gateway 3’ att primer sequence: 5`-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA-3`

Integrated DNA Technologies (IDT) synthesized constructs

Enhancer 2 minimal fragment and enhancer 1+5bp+2 fragment were synthesized by IDT and flanked by Gateway att sequences for recombination into the pg-lacZ.attB and pg-eya RB+3’UTR cDNA plasmids.

Enhancer 1+2 lacZ transcriptional reporter and cDNA fusion constructs

Enhancer 1 was amplified from the p-eya-enhancer 1.lacZ.attB plasmid with the following primers: 5`primer: 5`-ATA ATA AAG CTT ACT ACA CCT CGT ACC AAA TTC TCG G-3`and 3`primer: 5`-CCT GCT CAA CTC AAA TGG CCA GTT TCG TCT CC-3`Enhancer 2 was amplified from the p-eya enhancer 3.lacZ.attB plasmid using the following primers: 5’ primer: 5`-GGA GAC GAA ACT GGC CAT TTG AGT TGA GCA GG-3`and 3’ primer: 5`-ATA ATA GGT ACC TCA ACT GAT TCG ACT TGG TCG-3`PCR products were combined together using Gibson Assembly (New England Biolabs). Gateway recombination sequences were then added to the 5`and 3`ends of the product using the following primers: 5`primer: 5`-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC AAC ACT ACA CCT CGT ACC AAA TTC TCG G-3`and 3`primer: 5`-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA TCA ACT GAT TCG ACT TGG TCG AAA AGC-3`The resulting fragment (enhancer 1+2) was cloned into the pDONR201 plasmid and shuttled into pg-lacZ.attB and pg-eyaRB+3’UTR cDNA.attB plasmids using Gateway recombination cloning (Life Technologies).

Enhancer 1+spacer+2 lacZ transcriptional reporter

Enhancer 1 minimal fragment was amplified from the pg-eya enhancer 1 minimal.lacZ.attB plasmid using the following primers: 5`primer: 5`-AAA TAT TTG GAT ATG TGG GGG AAA GGG-3`and 3’ primer: 5`-ATA ATA GAA TTC GGC CAG TTT CGT CTC CTC TTT TGC-3`(adds an EcoRI site). The spacer fragment was amplified from the pg-eya intron 1-1.lacZ.attB plasmid using the following primers: 5`primer: 5`-ATA ATA GAA TTC TGA AAG ATC TCA ATT AGC TAA CCG-3`(adds an EcoRI site) and 3’ primer: 5`-ATA ATA TCT AGA CAA CTG CTA CCA TTT TGG CCA TTT C-3`(adds a XbaI site). Enhancer 2 was amplified from the p-eya enhancer #2.lacZ.attB plasmid using the following primers: 5’ primer: 5`-ATA ATA TCT AGA ATT TGA GTT GAGCAGGTCAGTTAATATTAC-3`(adds a XbaI site) and 3’ primer: 5`-TCA ACT GAT TCG ACT TGG TCG-3`The three fragments were ligated together to generate 1+spacer+2. The following primers were then used to amplify this product, which was then cloned into the p-lacZ.attB plasmid as a HindIII-KpnI fragment. 5’ primer: 5`-ATA ATA AAG CTT AAA TAT TTG GAT ATG TGG GGG AAA GGG-3`(adds a HindIII site) and 3’ primer: 5`-ATA ATA GGT ACC TCA ACT GAT TCG ACT TGG TCG-3`(adds a KpnI site).

Enhancer 1+spacer+2 eya RB cDNA fusion

The pg-eya RB cDNA+3’UTR.attB plasmid (see above) was digested with HindIII and KpnI resulting in a plasmid missing the Gateway cassette, hsp70 promoter and a portion of the eya RB cDNA. Into this plasmid was cloned the 1+spacer+2 region (see above) as a HindIII-KpnI fragment resulting in a p-eya 1+spacer+2 eya RB cDNA+3’UTR(partial).attB plasmid that is still missing the hsp70 minimal promoter and a portion of the eya RB cDNA. These pieces were amplified as a single fragment from pg-eya RB cDNA+3’UTR.attB using the following primers: 5’ primer: 5`-TCG AAT CAG TTG AGG TAC CTC TAG AGC-3`(adds a KpnI site) and 3’ primer: 5`-CCA GAG CCG GCG GTA CCC ACA CTG-3`(adds a KpnI site). This fragment was cloned into p-eya 1+spacer+2 eyaRB cDNA+3’UTR (partial).attB as a KpnI fragment to yield the final p-eya 1+spacer+2 eyaRB cDNA+3’ UTR.attB plasmid.

Cloning of enhancers into the promoterless pg-lacZ.attB vector

Enhancer 2 and the composite enhancer were amplified from the p-eya enhancer 2.lacZ.attB and p-eya composite enhancer.lacZ.attB plasmids respectively (primer sequences are listed in S2 Table). The 3’ primer adds 40bp of genomic sequence downstream of enhancer 2 and the transcriptional start site to ensure the entire endogenous promoter region was included. These 40bp were omitted from the above plasmids since a hsp70 minimal promoter is included within the plasmid. Gateway recombination sequences were added to the ends of each construct and the fragments were cloned into the pg-lacZ.attB plasmid that lacks a hsp70 promoter (Konrad Basler, University of Zurich, Switzerland) using standard Gateway Recombination Cloning.

Generation of transgenic fly strains

All lacZ reporter and cDNA fusion constructs were stably integrated into the pBAC(y+-attP-3B)VK00033 third chromosome landing site using PhiC31-mediated integration. Proper site-specific integration was confirmed by PCR with attP/attB primers and the correct sequence of the construct was confirmed. The composite enhancer-lacZ construct was also inserted into a second landing site on the third chromosome for comparison: PBac(y⁺-attP-9A)VK00019.

Molecular analysis of the eya¹ deletion

The genomic region surrounding the eya¹ deletion [31] was amplified from genomic DNA of the eya¹ stock (BL-3631). Genomic DNA from the same region was amplified from w¹¹¹⁸. The following primers were used to amplify the area surrounding the deletion: 5`primer: 5’-TTC CCG CTG GTG ACT TAC TG-3’ and 3`primer: 5’-GTT GTG AGG GAG CTG TCT GG-3’ The 5’ primer sits 2683 bp upstream of the eya RB transcriptional start site and the 3’ primer sits 702bp into the first intron. Q5 high-fidelity DNA polymerase (New England Biolabs) was used for the amplification. The PCR product was purified using GeneJet PCR Purification Kit (Thermo-Fisher #K0701). The amplified region from w¹¹¹⁸ is approximately 4kb while it is just over 2kb in the eya¹ mutant stock. Twelve sequencing primers were used to sequence the amplified genomic region in both directions. Primer sequences are listed within S3 Table. The eya¹ deletion is 1826bp in size: it begins 581bp upstream of the 5’ start of the eya² deletion and extends 344bp into the 5’ UTR of the eyaRB transcript. There are an additional 11bp that do not correspond to the published genomic region. BL = Bloomington Drosophila Stock Center

qPCR

qPCR was performed as previously described [49]. For each experiment three biological replicates were analyzed once. For each biological replicate, approximately 50 eye-antennal imaginal discs from wandering 3^rd instar larvae were dissected in PBS and immediately placed into a microcentrifuge tube containing 200ul of RLT buffer with β-mercaptoethanol (Qiagen #79216). The tissue was disrupted with a pestel for 1 minute. After disruption an additional 150ul of RLT buffer with β-mercaptoethanol was added to the tube and the sample homogenized using a QIAshredder column (Qiagen #79654). After homogenization total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen #74101). 100-200ng of total RNA was reverse transcribed to cDNA using the SuperScript III First Strand Synthesis System with oligo(dT) primers (Invitrogen). qPCR was performed on a Roche LightCycler 480 using SYBR Green I Master Mix (Roche). For each experiment, target genes were analyzed on biological triplicate samples and normalized to rp49. 3–4 serial dilutions of pooled cDNA were used to determine primer amplification efficiencies for each target gene. In eya¹ and eya² rescue experiments, primers specific to the endogenous eya RA and eya RB transcripts were used. Roche LightCycler 480 Software (Version 1.5) was used to calculate cycle threshold values and melting curves for each reaction. Relative expression and standard error was calculated using Relative Expression Software Tool (REST) [50]. Error bars generated by REST analysis reflect standard error determined by a confidence interval centered on the median, allowing representation of asymmetric tendencies in the data. Primers were designed using A plasmid Editor (ApE) or Fly Primer Bank [51]. Primer sequences are listed in S4 Table.

So binding site sequence analysis

Examination of the eya locus (both strands) for predicted So binding sties was performed using the following reported sequences: GTAANYNGANAYC [52], GTAANYNGANAYG [52], GGTATCA [53], GATATCA [53], TGATAC [54], TGATAC [32], CGATAC [32], ATTGATATCAAT [55], and TTGATATCAA [55].