Functional Dissection of Eyes absent Reveals New Modes of Regulation within the Retinal Determination Gene Network

Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Molecular and Cellular Biology (Impact Factor: 4.78). 10/2003; 23(17):5989-99. DOI: 10.1128/MCB.23.17.5989-5999.2003
Source: PubMed


The retinal determination (RD) gene network encodes a group of transcription factors and cofactors necessary for eye development. Transcriptional and posttranslational regulation of RD family members is achieved through interactions within the network and with extracellular signaling pathways, including epidermal growth factor receptor/RAS/mitogen-activated protein kinase (MAPK), transforming growth factor beta/DPP, Wingless, Hedgehog, and Notch. Here we present the results of structure-function analyses that reveal novel aspects of Eyes absent (EYA) function and regulation. We find that the conserved C-terminal EYA domain negatively regulates EYA transactivation potential, and that GROUCHO-SINE OCULIS (SO) interactions provide another mechanism for negative regulation of EYA-SO target genes. We have mapped the transactivation potential of EYA to an internal proline-, serine-, and threonine-rich region that includes the EYA domain 2 (ED2) and two MAPK phosphorylation consensus sites and demonstrate that activation of the RAS/MAPK pathway potentiates transcriptional output of EYA and the EYA-SO complex in certain contexts. Drosophila S2 cell two-hybrid assays were used to describe a novel homotypic interaction that is mediated by EYA's N terminus. Our data suggest that EYA requires homo- and heterotypic interactions and RAS/MAPK signaling responsiveness to ensure context-appropriate RD gene network activity.

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    • "The phosphatase activity is located at the C-terminus of the gene, which is also involved in interacting with So and Dac (Pignoni et al. 1997; Chen et al. 1997). The transactivator domain lays at the N-terminus, containing a proline-serine-threonine-rich region (Silver et al. 2003). The vertebrate Eya2 can rescue the eye-specific eya null mutant phenotype, causing eyeless flies and thus revealing a high degree of conservation in the eya gene sequence (Bonini et al. 1997). "
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    ABSTRACT: The compound eye of Drosophila melanogaster consists of hundreds of stereotypically organized unit eyes called ommatidia. The development of the eye begins in the fly embryo with the formation of the precursor cells of the presumptive eye-antennal imaginal disc. The disc expresses genes that belong to a so-called retinal determination network (RDN). The interplay between the genes of the RDN specifies and directs the development of the adult eye. The RDN includes highly conserved genes and integrate canonical signalling pathways, yet the outcome is totally distinctive from a typical vertebrate lens eye. The members of the RDN are highly interconnected forming complex, non-hierarchal loops controlling the transcription of one another. Knowledge gained from studying the RDN has helped us to better understand important developmental processes, such as cell specification and tissue growth. Astonishingly it has also demonstrated that eyes across species and phyla, albeit so different, share a common origin. In this chapter we will summarize the current knowledge of the RDN with its members and their interactions. In addition, we will briefly introduce the early specification steps of the eye-antennal imaginal disc in the embryo and later in the larvae. © 2013 Springer Science+Business Media New York. All rights are reserved.
    Full-text · Article · May 2013
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    • "cDNA-based studies have also suggested that an internal proline-serine-threonine-rich (PST) domain in Eya is required for transcriptional activation in cell culture reporter assays and for efficient induction of ectopic eyes in vivo [36]. Consistent with these results, our genomic rescue assays also show that the PST domain is required for Drosophila survival as well as normal eye development (data not shown; to be reported elsewhere). "
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    ABSTRACT: 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.
    Full-text · Article · Mar 2013 · PLoS ONE
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    • "eyes absent (eya) encodes a highly conserved transcriptional coactivator and protein phosphatase whose homologs play vital roles in human development [1], [2], [3], [4]. Mutations in human EYA1 lead to the autosomal dominant disorder known as branchio-oto-renal (BOR) syndrome, characterized by craniofacial anomalies, hearing loss, and kidney defects [5]. "
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    ABSTRACT: Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that plays an essential role in eye development and survival in Drosophila. Ectopic eye induction assays using cDNA transgenes have suggested that mitogen activated protein kinase (MAPK) activates Eya by phosphorylating it on two consensus target sites, S402 and S407, and that this activation potentiates the ability of Eya to drive eye formation. However, this mechanism has never been tested in normal eye development. In the current study, we generated a series of genomic rescue transgenes to investigate how loss- and gain-of-function mutations at these two MAPK target sites within Eya affect Drosophila survival and normal eye formation: eya(+)GR, the wild-type control; eya(SA)GR, which lacks phosphorylation at the two target residues; and eya(SDE)GR, which contains phosphomimetic amino acids at the same two residues. Contrary to the previous studies in ectopic eye development, all eya genomic transgenes tested rescue both eye formation and survival equally effectively. We conclude that, in contrast to ectopic eye formation, MAPK-mediated phosphorylation of Eya on S402 and S407 does not play a role in normal development. This is the first study in Drosophila to evaluate the difference in outcomes between genomic rescue and ectopic cDNA-based overexpression of the same gene. These findings indicate similar genomic rescue strategies may prove useful for re-evaluating other long-standing Drosophila developmental models.
    Full-text · Article · Dec 2012 · PLoS ONE
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