Gene name - eyes absent
Cytological map position - 26E1--26E4
Symbol - cli
Genetic map position - 2-17
Classification - protein tyrosine phosphatase, eyes absent family
Cellular location - nuclear
The eyes absent gene, recently and more correctly termed clift, is required at an early stage in development of the compound eye. eya is not expressed in the embryonic eye primordia or in eye discs during the first larval instar, suggesting that unlike eyeless, eya is not involved in determining early commitment to eye cell fate. In fact the newly discovered mammalian eya homolog has been shown to be regulated by Pax6, the vertebrate homolog of Drosophila Eyeless (Xu, 1997). In Drosophila eya mutants, progenitor cells in the eye disc undergo programmed cell death anterior to the morphogenetic furrow, rather than proceeding along the developmental pathway leading to retinal differentiation. Thus the absence of eyes is not due to a lack of precursors but to death of those precursors. A low level of cell death normally occurs at this stage, suggesting that eya activity influences the balance between cellular differentiation and death, that is, Eya has a cell survival function. Molecular analysis identifies Eya as nuclear protein expressed in progenitor cells prior to differentiation. It thus appears that eyes absent activity is required for the survival of eye progenitor cells at a critical stage in morphogenesis (Bonini, 1993).
Besides functioning as a transcriptional co-activator that functions in conjunction with Dachshund and Sine oculis, Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceeds by means of a thiol-phosphate intermediate. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. The phosphatase function of Eya switches the function of Six1f-Dach, Eya interactors in mammals, from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival (Li, 2003; Rayapureddi, 2003; Tootle, 2003).
The fly eyes absent (eya) gene, essential for compound eye development in Drosophila, has been shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, has been defined: eya is essential for eyeless directed eye formation (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). Directing eyeless expression to discs that generate legs, wings and the antennal region of the head generates ectopic eyes in these regions. Eya is ectopically expressed in regions where eyeless directs ectopic eye formation. eya is essential for these ectopic eyes as ectopic eyes fail to form in eya mutants. eya can itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Directing ectopic eya expression to imaginal discs induces ectopic eyeless gene expression in the antennal region of the eye-antennal disc but fails to induce ectopic eyeless expression in other discs, even though ectopic ommatidia are formed and ectopic expression of Glass occurs. eya and eyeless together are more effective in eye formation than either gene alone: when expressed together, ectopic eyes are larger and form with higher penetrance than is the case with either eyeless or eya alone; expressed togther, eye formation occurs on the genitalia, a condition never observed in individuals with either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation (Bonini, 1997).
eyes absent also plays a role in the embryonic determination of somatic gonadal precursor (SGP) cell fate. The gonad forms from cells of two lineages: the germline and soma (see gonadogenesis and oogenesis for more information). The somatic gonadal cells generate the various cell types within the testis or ovary that support gametogenesis.
It may be useful here to provide a brief description of the origin of SGP cells: each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. Using markers for the precursors of the somatic cells of the gonad, five discrete steps have been identified in gonadal development:
The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior gonadal precursor fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. The proper regional identities, as established by homeotic gene function, are required for the arrest of migration at the correct position. abd A is required in a population of cells within parasegments 10 and 11 that partially ensheath the coalescing gonad. Mutations in iab-4, a distal enhancer element, abolish expression of abd A within these cells, blocking the coalescence of the gonad (Boyle, 1995).
clift has been identified as a regulator of Drosophila gonadogenesis. When cloned, clift turned out to be identical to eyes absent. Mutations in clift abolish gonad formation and produce female sterility. In addition to showing an eye and gonad phenotype, some clift alleles show reduced or absent ocelli and abnormal morphology of the adult brain (Nusslein-Volhard, 1984). Just as with abdominal A, clift is expressed within SGP as these cells first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in gonadogenesis. In abdominal A mutants, clift fails to be expressed, and in abd A overexpressors, clift is expressed ectopically. Despite the early expression of clift, SGP's are specified in the absence of clift function. However, they fail to maintain their fate. Consequently, germ cells do not coalesce into a gonad. In addition, using clift as a marker, it has been shown that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with a gene regulatory hierarchy involving abd A within the mesoderm. While loss of wg abolishes gonadal precursors, ectopic expression expands the population such that most cells within lateral mesoderm adopt gonadal precursor fates. Initial dorsoventral positioning of somatic gonadal precursors relies on a regulatory cascade that establishes dorsal fates within the mesoderm. tinman appears to mediate the role of ectodermally expressed decapentaplegic; in tinman mutants few or no SGP cells are detected. clift expression is subsequently refined through negative regulation by bagpipe, a gene that specifies nearby visceral mesoderm. Thus, these studies identify essential regulators of gonadal precursor specification and differentiation and reveal novel aspects of the general mechanism whereby somatic gonadal cell fate is allocated within the mesoderm (Boyle, 1997).
Does clift/eyes absent have the same targets in the eye that it has in somatic gonad precursors? Is there a unified role for this gene in the two organs? In the eye, eyes absent is implicated in cell survival, while in the gonads clift is implicated in gonadal cell fate. One clift mutant, which acts as a strong allele in gonadal fate determination, nevertheless produces a transcript. Use of this mutant allowed an examination of SGP cell development in the absence of clift function. Shortly after SGP cells are specified in cli mutants there is a reduction in the number of cli expressing SGP cells, such that by germ band retraction, few SGP cells remain. The few cells that remain fail to complete their migration to the position of gonad formation (Boyle, 1997). It would therefore appear that there might be a role for clift in SGP survival in the mesoderm as there is for eya in photoreceptor precursor survival in the eye. The answer to whether there is a unified role for eya/clift in eyes and in the mesoderm awaits discovery of the target(s) for this gene.
Bases in 5' UTR - 395 for type 1 and 210 for type II
Exons - 5 for each transcript
Bases in 3' UTR - 553
There are two identified cDNA sequences initiating from different promoters. The first 19 amino acids of the type I cDNA and the first 25 of the type II are generated by alternative splicing; these transcripts differ in their first exons (Bonini, 1993).
Comparison of the vertebrate proteins with Drosophila Eya reveals several domains of homology. The largest domain is the C-terminal portion of the protein, referred to as ED1 (for Eya homology domain1), which shows 53% identity over 271 amino acids in all the vertebrate and fly clones. The vertebrate ED1 sequences are more similar to one another than to the fly's ED1 sequence. Within the N-terminal domain, the sequences show little conservation. A short region of weak homology, ED2, occurs between the vertebrate and fly sequences in the amino terminus. In this domain, a run of spaced tyrosine residues is conserved. The fly sequence has a weak PEST protein degredation sequence; in a similar location, the mouse Eya2 sequence has a region that may serve as a weak PEST site. The fly Eya protein sequence has a consensus nuclear localization sequence at the amino terminus, and the protein is nuclear by immunocytochemistry. However, none of the vertebrate sequences show a motif indicative of a nuclear localization sequence (Zimmerman, 1997). The Drosophila protein has several potential cyclic nucleotide-dependent, protein kinase C and tyrosine kinase phosphorylation sites (Bonini, 1993)
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. A structure-function analyses reveals novel aspects of Eyes absent (Eya) function and regulation. The conserved C-terminal Eya domain negatively regulates Eya transactivation potential, and Groucho-Sine oculis (So) interactions provide another mechanism for negative regulation of Eya-So target genes. The transactivation potential of Eya maps 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. These data suggest that EYA requires homo- and hetero-typic interactions and RAS/MAPK signaling responsiveness to ensure context-appropriate RD gene network activity (Silver, 2003).
date revised: 20 November 2003
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