Eyes absent tyrosine phosphatase activity is not required for Drosophila development or survival.

Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context

Animals rescued with <i>eya*GR</i> have normal photoreceptor axon projections.

<p>Third instar eye-brain complexes from <i>Df/+; ro-lacZ^tau/+</i> (A,D,G), <i>eya^cli/Df; eya⁺GR/ro-lacZ^tau</i> (B,E,H), and <i>eya^cli/Df; eya^NQGR/ro-lacZ^tau</i> (C,F,I) stained with anti-Chaoptin and anti-β-galactosidase. (A–C) Projections from photoreceptors R1–8 are visualized by using mAb24B10 (anti-Chaoptin). (D–F) Projections from photoreceptors R2–R5, which terminate in the lamina, are revealed using <i>ro-lacZ^tau</i> (anti-β-galactosidase). (G-I) Merge of channels. <i>eya^NQGR</i> rescued animals show normal photoreceptor axon projections, similar to flies rescued with <i>eya⁺GR</i> and <i>Df/+; ro-lacZ^tau/+</i>. (J) 30–40 eye-brain complexes were scored for overshooting axon bundles by investigators who were blind to the genotype. The average number of overshooting axon bundles per brain is the same for <i>Df/+; ro-lacZ^tau/+</i>, <i>eya^cli/Df; eya⁺GR/ro-lacZ^tau</i> and <i>eya^cli/Df; eya*GR/ro-lacZ^tau</i>. n, number of brains scored. Error bars indicate standard deviation.</p

Dynamic Rewiring of the Drosophila Retinal Determination Network Switches Its Function from Selector to Differentiation

Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the "retinal determination gene network," composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development.status: publishe

Dynamic rewiring of the Drosophila retinal determination network switches its function from selector to differentiation.

Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the "retinal determination gene network," composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development

Flies carrying one copy of <i>eya*GR</i> show normal viability.

<p>Progeny from <i>w; eya^cli/CyO; eya*GR</i>×<i>w; Df/CyO</i> cross are present at Mendelian ratios, indicating that rescued files carrying a single copy of <i>eya*GR</i> do not have a survival disadvantage compared with their heterozygous siblings. χ² critical (1 d.f. p 0.05) = 3.84.</p

<i>eya*GR</i> constructs rescue larval eye imaginal disc development.

<p>Immunohistochemical detection of Eya (A–C) and the pan-neural marker Elav (D–F) in <i>Df/+</i> (A,D), <i>eya^cli/Df; eya⁺GR/+</i> (B,E) and <i>eya^cli/Df; eya^NQGR/+</i> (C,F) third instar larval eye imaginal discs. Expression patterns and levels of Eya and Elav are indistinguishable among <i>eya</i> null eye discs carrying one copy of <i>eya⁺GR</i>, <i>eya^NQGR</i> and <i>Df</i> heterozygotes.</p

Mutations in the tyrosine phosphatase active site of Eya do not affect response to light.

<p>ERG recordings from wild-type <i>Canton S</i> (A), <i>eya^cli/Df; eya⁺GR/+</i> (B) and <i>eya^cli/Df; eya^NQGR/+</i> (C) 3-day-old retinas show similar curves and suggest rescued animals have a normal light response.</p

Schematic of the <i>eya⁺GR</i> transgene and <i>eya</i> locus with the position of mutations indicated.

<p>A 45.5 kb region of the genomic DNA surrounding the <i>eya</i> locus (shown as a red bar) was recombineered into <i>attB-P[acman]-Ap^R</i>. <i>eya</i> has three alternative transcripts (<i>eya-RA</i>, <i>-RB</i>, and <i>-RC</i>). Red 4-point stars indicate the tyrosine phosphatase active site mutations; D493N and E728Q are in the 3^rd and 5^th exons of <i>eya</i>, respectively.</p

<i>eya*GR</i> constructs restore eye development and viability in <i>eya</i> mutants.

<p>(A) <i>eya²</i> homozygous adults completely lack eyes. <i>eya²</i> adults rescued with one copy of <i>eya⁺GR</i> (C,H) or <i>eya^NQGR</i> (E,J) show similar external and internal eye morphology compared with wild-type <i>Canton S</i> (B,G). A single copy of <i>eya⁺GR</i> (D,I) or <i>eya^NQGR</i> (F,K) can fully rescue eye morphology and survival in <i>eya^cli/Df</i> flies, which have no endogenous <i>eya</i> function.</p

Adults rescued with <i>eya*GR</i> show normal fertility.

<p>Fertility assays of animals of the indicated sex and genotypes show both males and females carrying one copy <i>of eya*GR</i> have normal fertility. No statistically significant difference was observed compared to control <i>eya^cli/CyO</i> and <i>Df/CyO</i> flies using ANOVA. Error bars indicate standard deviation.</p