The eyes absent-like genes encode a group of putative transcriptional coactivators with a sole representative in Drosophila and several members in mammals. Haploinsufficiency of the human EYA1 gene results in branchio-oto-renal syndrome characterized by developmental anomalies of the branchial arches, the three compartments of the ear and the kidney. As a first step towards a functional analysis of this gene in lower vertebrates, its zebrafish homolog, eya1, was isolated and its expression pattern during embryogenesis studied. The eya1 cDNA predicts a protein with 84.7% identity with the human homolog. Transcripts are first detected at the tailbud stage in presumptive cranial placodal precursor cells. Thereafter, eya1 expression continues in anterior pituitary, olfactory, otic, and lateral line placodes. Aside from these placodal sites of expression, eya1 transcripts are observed in the somites, developing pectoral fins, and branchial arches. No expression is found in pronephros or the Wolffian duct of the zebrafish renal system. Within the developing ear, eya1 expression becomes confined to the ventral part of the otic vesicle from where the acoustic ganglion precursor cells arise and the sensory patches differentiate. In the lateral line, eya1 is expressed in the placodes, ganglia, migrating primordia, and receptive organs at all developmental stages, including both the differentiating hair and supporting cells. Taken together, these results indicate a remarkable similarity in both the structure and expression pattern of eya1 between higher and lower vertebrates, suggesting that the function of this gene has been conserved throughout vertebrate evolution (Sahly, 1999).

Three members of a new family of vertebrate genes, designated Eya1, Eya2 and Eya3 have been identified that share high sequence similarity with the Drosophila eyes absent gene. Comparison of all three murine Eya gene products with that encoded by the Drosophila eya gene defines a 271 amino acid carboxyl terminal Eya domain, apparently one that has been highly conserved during evolution. Eya1 and Eya2, which are closely related, are extensively expressed in cranial placodes, in the branchial arches and CNS and in complementary or overlapping patterns during organogenesis. Eya3 is also expressed in the branchial arches and CNS, but lacks cranial placode expression. All three Eya genes are expressed in the developing eye. Eya1 is expressed in developing anterior chamber structures, including the lens placode, the iris and ciliary region and the prospective corneal ectoderm. Eya1 is also expressed in retinal pigment epithelium and optic nerve. Eya2 is expressed in neural retina, sclera and optic nerve sheath. Moreover, Eya1 and Eya2 expressions in the lens and nasal placode overlap with and depend upon expression of Pax6 (Drosophila homolog: Eyeless). The high sequence similarity with Drosophila eya, the conserved developmental expression of Eya genes in the eye and the Pax6 dependence of Eya expression in the lens and nasal placode indicates that these genes likely represent functional homologs of the Drosophila eya gene. These results suggest that members of the Eya gene family play critical roles downstream of Pax genes in specifying placodal identity and support the idea that despite enormous morphological differences, the early development of insect and mammalian eyes is controlled by a conserved regulatory hierarchy (Xu, 1997a).

Two human and two mouse homologs of the fly eyes absent gene have been identified. Sequence comparison reveals a large domain of approximately 270 amino acids in the carboxyl terminus of the predicted mammalian proteins that shows 53% identity between the fly sequence and all of the vertebrate homologs. This Eya-homology domain is of novel sequence, with no previously identified motifs. RNA hybridization studies indicate that the mouse genes are expressed during embryogenesis and in select tissues of the adult. Both mouse Eya genes are expressed in the eye, suggesting that these genes may function in eye development in vertebrates as eya does in the fly. The mouse Eya2 gene maps to chromosome 2 in the region syntenic with human chromosome 20q13, and the mouse Eya2 gene maps to chromosome 4 in the region syntenic with human chromosome 1p36. These findings support the notion that several families of genes (Pax-6/eyeless, Six-3/sine oculis, and Eya) play related and critical roles in the eye for both files and vertebrates (Zimmerman, 1997).

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified, but the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis remain largely unknown. The eyes absent (eya) gene of Drosophila encodes a novel nuclear protein of unknown molecular function. Eya1 and Eya2, two mouse homologs of Drosophila eya, are expressed initially during limb development in connective tissue precursor cells. Later in limb development, Eya1 and Eya2 expression is associated with cell condensations that form different sets of limb tendons. Eya1 expression is largely restricted to flexor tendons, while Eya2 is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that Eya genes participate in the patterning of the distal tendons of the limb. The ability of the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1-3 to function as transactivation domains was examined in order to investigate the molecular functions of the Eya gene products. Eya gene products can act as transcriptional activators and support a role for this molecular function in connective tissue patterning (Xu, 1997b).

Planarians can regenerate any missing body part, requiring mechanisms for the production of organ systems in the adult, including their prominent tubule-based filtration excretory system called protonephridia. This study identified a set of genes, Six1/2-2, POU2/3, hunchback, Eya and Sall, that encode transcription regulatory proteins that are required for planarian protonephridia regeneration. During regeneration, planarian stem cells are induced to form a cell population in regeneration blastemas expressing Six1/2-2, POU2/3, Eya, Sall and Osr that is required for excretory system formation. POU2/3 and Six1/2-2 are essential for these precursor cells to form. Eya, Six1/2-2, Sall, Osr and POU2/3-related genes are required for vertebrate kidney development. Planarian and vertebrate excretory cells express homologous proteins involved in reabsorption and waste modification. Furthermore, novel nephridia genes were identified. These results identify a transcriptional program and cellular mechanisms for the regeneration of an excretory organ and suggest that metazoan excretory systems are regulated by genetic programs that share a common evolutionary origin (Scimone, 2011).

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. The role of Eya1 was examined during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures fail to form in Eya1^-/- embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1^-/- mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1^-/- embryos, indicating that Six1 expression in those structures is Eya1 dependent. In Eya1^-/- embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. The surface ectoderm of the 3rd and 4th pharyngeal regions shows increased cell death at E10.5 in Eya1^-/- embryos. These results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid (Xu, 2002).

To understand the molecular basis of sensory organ development and disease, the zebrafish mutation dog-eared (dog), which is defective in formation of the inner ear and lateral line sensory systems, was cloned and characterized. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders (Kozlowski, 2005).

To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, eya4 expression was studied during zebrafish development and eya4 deficiency was characterized. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4 focused analyses on atp1b2b, which encodes the beta2b subunit of the zebrafish Na+/K+-ATPase. atp1b2b levels were demonstrated to be reduced in eya4 morphant fish, and morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. It is concluded that eya4 regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish (Wang, 2008).

A novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 has been identified. Dach2, is expressed in the developing somite prior to any myogenic genes, with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context -- myogenesis rather than eye development -- and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6 (Heanue, 1999).

Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologs of these genes, Six and Eya, respectively, show overlapping expression patterns during development. It has been hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters is observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, the intracellular distribution of Six and Eya proteins were examined in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induces nuclear translocation of Eya1, Eya2, and Eya3, which are otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 does not result in nuclear localization of any Eya proteins. Six and Eya proteins coimmunoprecipitate from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, are necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, is not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 is observed by yeast two-hybrid analysis. These results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes (Ohto, 1999).

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, the molecular interactions among the encoded proteins was examined. Eya interacts directly with Six but never with Dach. Dach transactivates a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach is repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template is dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. These results demonstrate that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis (Ikeda, 2002).

Eya1 encodes a transcriptional co-activator and is expressed in cranial sensory placodes. It interacts with and functions upstream of the homeobox gene Six1 during otic placodal development. Their role in cranial sensory neurogenesis was examined. The data show that the initial cell fate determination for the vestibuloacoustic neurons and their delamination appears to be unaffected in the absence of Eya1 or Six1 as judged by the expression of the basic helix-loop-helix genes, Neurog1, which specifies the neuroblast cell lineage, and Neurod, which controls neuronal differentiation and survival. However, both genes are necessary for normal maintenance of neurogenesis. During the development of epibranchial placode-derived distal cranial sensory ganglia, while the phenotype appears less severe in Six1 than in Eya1 mutants, an early arrest of neurogenesis was observed in the mutants. The mutant epibranchial progenitor cells fail to express Neurog2, which is required for the determination of neuronal precursors, and other basic helix-loop-helix as well as the paired homeobox Phox2 genes that are essential for neural differentiation and maintenance. Failure to activate their normal differentiation program results in abnormal apoptosis of the progenitor cells. Furthermore, disruption of viable ganglion formation leads to pathfinding errors of branchial motoneurons. Finally, these results suggest that the Eya-Six regulatory hierarchy also operates in the epibranchial placodal development. These findings uncover an essential function for Eya1 and Six1 as critical determination factors in acquiring both neuronal fate and neuronal subtype identity from epibranchial placodal progenitors. These analyses define a specific role for both genes in early differentiation and survival of the placodally derived cranial sensory neurons (Zou, 2004).