Differential requirements for the Pax6(5a) genes eyegone and twin of eyegone during eye development in Drosophila

In eye development the tasks of tissue specification and cell proliferation are regulated, in part, by the Pax6 and Pax6(5a) proteins respectively. In vertebrates, Pax6(5a) is generated as an alternately spliced isoform of Pax6. This stands in contrast to the fruit fly, Drosophila melanogaster, which has two Pax6(5a) homologs that are encoded by the eyegone and twin of eyegone genes. In this report we set out to determine the respective contributions that each gene makes to the development of the fly retina. Here we demonstrate that both eyg and toe encode transcriptional repressors, are expressed in identical patterns but at significantly different levels. We further show, through a molecular dissection of both proteins, that Eyg makes differential use of several domains when compared to Toe and that the number of repressor domains also differs between the two Pax6(5a) homologs. We predict that these results will have implications for elucidating the functional differences between closely related members of other Pax subclasses

Retinal Expression of the <i>Drosophila eyes absent</i> Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

<div><p>The <i>eyes absent</i> (<i>eya</i>) gene of the fruit fly, <i>Drosophila melanogaster</i>, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of <i>eya</i> leads to the complete elimination of the compound eye while forced expression of <i>eya</i> in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina <i>eya</i> is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. In this report we explore the mechanisms by which <i>eya</i> expression is spatially and temporally governed in the developing eye. We demonstrate that multiple cis-regulatory elements function cooperatively to control <i>eya</i> transcription and that spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, we show that the loss of <i>eya</i> expression in <i>sine oculis</i> (<i>so</i>) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis.</p></div

Eya protein is detected in non-transformed <i>so</i>^<i>3</i> mutant clones.

<p>(A-L) Eye discs and adult heads of the genotype: <i>eyflp; FRT42D so</i>^<i>3</i> <i>/FRT42D Ubi-GFP</i> that contain <i>so</i>^<i>3</i> mutant clones. (A-H) Light microscope images of developing eye-antennal discs. Yellow arrows identify <i>so</i>^<i>3</i> clones that have not transformed into head epidermis and still express <i>eya</i>. Green arrows demarcate large <i>so</i>^<i>3</i> clones that are transforming into head epidermis and lack Eya protein. Red = F-actin, green = GFP, blue = Eya. (I-L) SEM images of adult <i>Drosophila</i> 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 <i>so</i>^<i>3</i> clones were examined. Scale bar, 100μm.</p

The composite enhancer fully restores eye development to <i>eya</i>^<i>1</i> and <i>eya</i>^<i>2</i> mutants.

<p>(A) qRT-PCR quantification of <i>eya</i> 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 <i>Drosophila</i> compound eyes and heads from enhancer cDNA fusion rescue experiments. Each enhancer is driving expression of the <i>eya</i> RB isoform within the developing eye of <i>eya</i>^<i>1</i> and <i>eya</i>^<i>2</i> mutants. (B,I) <i>Enhancer 1—eya RB cDNA</i> fusion partially rescues 100% of animals examined. (C,J) <i>Enhancer E—eya RB cDNA</i> fusion partially rescues 100% of animals examined. Rescue efficiency is significantly reduced in <i>eya</i>^<i>1</i> background. (D,K) <i>Enhancer 2—eya RB cDNA</i> fusion does not rescue either <i>eya</i>^<i>1</i> or <i>eya</i>^<i>2</i> mutants. (E,L) <i>Composite enhancer—eya cDNA</i> fusion fully rescues 100% of animals examined to wild type eye size. (F,M) <i>Enhancer 1+E—eya cDNA</i> fusion partially rescues 100% of animals examined. (G,N) <i>Enhancer 3—eya cDNA</i> fusion does not rescue <i>eya</i>^<i>1</i> or <i>eya</i>^<i>2</i> mutants. (H,O) <i>Enhancer 4—eya cDNA</i> fusion does not rescue <i>eya</i>^<i>1</i> or <i>eya</i>^<i>2</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006462#pgen.1006462.t001" target="_blank">Table 1</a>. Scale bar, 100μm.</p

Cooperative interactions between enhancers 1 and E drive eye development.

<p>(A-K) Light microscope images of adult <i>Drosophila</i> compound eyes and heads from single and combination enhancer—<i>eya</i> RB cDNA fusion rescue experiments. Each enhancer is driving expression of the <i>eya</i> RB isoform within the developing eye of <i>eya</i>^<i>1</i> mutants. (A) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; extant enhancer E—eya RB cDNA</i>. Expression of the <i>eya</i> RB cDNA driven by the extant enhancer alone weakly rescues 100% of animals examined. (B) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 1—eya RB cDNA</i>. Expression of the <i>eya</i> RB cDNA driven by enhancer 1 alone partially rescues 100% of animals examined. (C) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 2—eya RB cDNA</i>. Expression of the <i>eya</i> RB cDNA driven by enhancer 2 alone does not rescue the no-eye phenotype. (D) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 3—eya RB cDNA</i>. Expression of the <i>eya</i> RB cDNA driven by enhancer 3 alone does not rescue the no-eye phenotype. (E) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 4—eya RB cDNA</i>. Expression of the <i>eya</i> cDNA driven by enhancer 4 alone does not rescue the no-eye phenotype. (F) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer PSE—eya RB cDNA</i>. Expression of the <i>eya</i> RB cDNA driven by the PSE enhancer alone does not rescue the no-eye phenotype. (G) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 1—eya RB cDNA/extant enhancer E—eya RB cDNA</i>. Combining <i>enhancer 1—eya RB cDNA</i> and <i>enhancer E—eya RB cDNA</i> constructs increases the quality of rescue as demonstrated by the larger eye size in 100% of animals examined. (H) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 2—eya RB cDNA/extant enhancer E—eya RB cDNA</i>. Combining <i>enhancer 2—eya RB cDNA</i> and <i>enhancer E—eya RB cDNA</i> constructs does not increase the quality of rescue over the extant enhancer alone. (I) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 3—eya RB cDNA/extant enhancer E—eya RB cDNA</i>. Combining <i>enhancer 3—eya RB cDNA</i> and <i>enhancer E—eya RB cDNA</i> constructs does not increase the quality of rescue over the extant enhancer alone. (J) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer 4—eya RB cDNA/extant enhancer E—eya RB cDNA</i>. Combining <i>enhancer 4—eya RB cDNA</i> and <i>enhancer E—eya RB cDNA</i> constructs does not increase the quality of rescue over the extant enhancer alone. (K) <i>eya</i>^<i>1</i><i>/eya</i>^<i>1</i><i>; enhancer PSE—eya RB cDNA/extant enhancer E—eya RB cDNA</i>. Combining <i>enhancer PSE—eya RB cDNA</i> and <i>enhancer E—eya RB cDNA</i> 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.</p

Retinal progenitors within <i>so</i>^<i>1</i> mutants progressively transform into head epidermis.

<p>(A-M) Light microscope images of developing wild type, <i>so</i>^<i>1</i>, and <i>so</i>^<i>1</i>, <i>eya composite enhancer GAL4</i>, <i>UAS-p35</i> eye-antennal discs. Green = Otd and red = Cut. Expression of both <i>otd</i> and <i>cut</i> is de-repressed within the eye field of <i>so</i>^<i>1</i> mutant discs over the course of larval eye development. Although cell death has been blocked in the eye disc of <i>so</i>^<i>1</i> mutants by the caspase inhibitor p35, expression of <i>otd</i> and <i>cut</i> 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.</p

Spacing between enhancers is required for <i>eya</i> expression and function.

<p>(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 <i>Drosophila</i> compound eyes and heads from enhancer—<i>eya</i> RB cDNA rescue experiments. Each enhancer is driving expression of the <i>eya</i> RB isoform within the developing eye of either <i>eya</i>^<i>1</i> or <i>eya</i>^<i>2</i> mutants. (P) SEM image of adult <i>Drosophila</i> compound eyes and heads from <i>enhancer 1+spacer+2—eya RB cDNA</i> rescue experiments of <i>eya</i>^<i>1</i> mutants. (A-C) Expression driven by enhancer 1+2 is only activated late in eye development in some <i>eya</i> expressing cells posterior to the furrow. (D) Expression driven by enhancer 1+2 is activated in very few cells in <i>eya</i>^<i>2</i> mutant discs. (E) The <i>enhancer 1+2—eya RB cDNA</i> does not rescue <i>eya</i>^<i>2</i> mutants. (F-H) Expression driven by enhancer 1+5bp+2 is activated mostly in non-<i>eya</i> expressing cells early in development while later activation is seen in <i>eya</i> 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 <i>eya</i>^<i>2</i> mutants. (J) The <i>enhancer 1+5bp+2—eya RB cDNA</i> does not rescue <i>eya</i>^<i>2</i> mutants. (K-M) Expression driven by enhancer 1+spacer+2 restores some early expression in <i>eya</i> expressing cells but does not fully recapitulate <i>eya</i> expression at all stages of development. (N) Expression driven by enhancer 1+spacer+2 is strongly activated throughout the eye disc of <i>eya</i>^<i>2</i> mutants. (O) <i>Enhancer 1+spacer+2—eya RB cDNA</i> partially rescues 100% of <i>eya</i>^<i>2</i> mutants suggesting a restoration of function. (P) <i>Enhancer 1+spacer+2—eya RB cDNA</i> partially rescues 100% of <i>eya</i>^<i>1</i> 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 <i>eya</i>^<i>2</i> discs. (S) The 319bp spacer does not rescue <i>eya</i>^<i>2</i> 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</p

Multiple enhancers control expression of <i>eya</i> in the developing eye.

<p>(A) Illustration of the genomic map of the <i>eya</i> 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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006462#pgen.1006462.ref020" target="_blank">20</a>]. E = 319bp extant enhancer previously identified in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006462#pgen.1006462.ref011" target="_blank">11</a>]. Enhancer 1 (IAM) = immediately ahead of morphogenetic furrow enhancer was previously identified and named by Graeme Mardon’s group in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006462#pgen.1006462.ref020" target="_blank">20</a>]. 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. <i>eya</i>^<i>1</i> and <i>eya</i>^<i>2</i> 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</p

<i>eya</i> retinal enhancers remain transcriptionally active in <i>so</i> mutants.

<p>(A-F) Light microscope images of wandering third instar <i>so</i>^<i>1</i> mutant eye-antennal discs. Each enhancer is driving expression of lacZ within <i>so</i>^<i>1</i> eye-antennal discs. LacZ reporter activation is detected with an antibody that recognizes the β-galactosidase enzyme. (A) <i>so</i>^<i>1</i><i>; enhancer 1—lacZ</i>. Reporter expression driven by enhancer 1 is activated only in cells at the far posterior edge of the eye disc. (B) <i>so</i>^<i>1</i><i>; enhancer E–lacZ</i>. Reporter expression driven by the extant enhancer E is weakly activated throughout the eye disc. (C) <i>so</i>^<i>1</i><i>; enhancer 2 –lacZ</i>. Reporter expression driven by enhancer 2 is strongly activated throughout the remaining eye disc. (D) <i>so</i>^<i>1</i><i>; enhancer 1+E+2 –lacZ</i>. Reporter expression driven by the composite enhancer is strongly activated throughout the eye disc. (E) <i>so</i>^<i>1</i><i>; enhancer 3 –lacZ</i>. Reporter expression driven by enhancer 3 is activated broadly throughout the entire eye-antennal disc. (F) <i>so</i>^<i>1</i><i>; enhancer 4 –lacZ</i>. 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 <i>so</i>^<i>3</i> null clones have been generated. The absence of GFP marks the position of clones lacking <i>so</i>. Green = GFP, red = Eya protein, blue = lacZ. Yellow arrows mark the position of <i>so</i>^<i>3</i> 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.</p

Eya expression is lost progressively in <i>so</i>^<i>1</i> mutants as a result of increased cell death in retinal progenitors.

<p>(A-O) Light microscope images of developing <i>so</i>^<i>1</i> mutant eye discs. (A-E) Green arrows indicate regions containing Eya protein. The amount of Eya protein is progressively lost in <i>so</i> mutant retinas. (F-O) Light microscope images of developing <i>so</i>^<i>1</i> 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 <i>so</i>^<i>1</i> mutants is elevated in both progenitor and precursor cells. (P-Q) Light microscope images of developing <i>so</i>^<i>1</i>, <i>eya composite enhancer GAL4</i>, <i>UAS-p35</i>. Cell death has been blocked in the eye disc of <i>so</i>^<i>1</i> mutants by expression of the caspase inhibitor p35. Blocking death does not prevent the loss of <i>eya</i> expression in late third instar <i>so</i>^<i>1</i> 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.</p