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The human EYA1 gene is an ortholog of the fruit fly eyes absent gene (eya), which was identified as a regulator of compound eye development. In contrast to the single eya gene found in Drosophila melanogaster, mammals possess four Eya paralogs, designated Eya1-4. The respective gene products are characterized by a C-terminal domain called the Eya domain, which is conserved in both length (271 to 274 amino acids [aa]) and sequence. The Eya domain has an intrinsic phosphatase activity (24, 39, 53) and is required for protein-protein interactions (38). Recent studies provided evidence that the phosphatase function of Eya is involved in the innate immune system and the regulation of DNA damage response (8, 35). Interestingly, all Eya interaction partners that have been identified so far, for example, the Six and Dach proteins or inhibitory G protein α (Gα) subunits, bind to the Eya domain (5, 10, 34). It has been demonstrated that the cooperative action of Eya and Six is essential for the development of several tissues and organs in a variety of species throughout evolution (reviewed in reference 6). In vertebrates, Six induces nuclear translocation of Eya and recruitment to the DNA, and Eya enhances Six-mediated activation of target gene expression (34). The N termini of Eya proteins, which are highly divergent between Eya family members, harbor a proline-serine-threonine (PST)-rich transactivation domain, which is indispensable for their function as coactivators of transcription (31, 45, 50, 58). Natural target genes of the vertebrate Eya-Six complex are, for example, Six2, Sall1, and Myogenin. Activation of Six2 and Sall1 expression has been shown to be essential for proper kidney development in the mouse, whereas activation of Myogenin is required for muscle development (3, 4, 46). Mutations in the human EYA1 gene are associated with several congenital disorders, like BOR (branchio-oto-renal) and BO (branchio-oto) syndrome, as well as ocular defects. BOR syndrome patients suffer from severe malformations of the ears, the branchial arch derivatives, and the kidneys, while in BO syndrome patients, the kidneys are not affected (11, 12, 30). The physiological importance of Eya1 was underlined by knockout studies in mice. It was shown that the phenotype of Eya1-heterozygous mice resembles the characteristic symptoms of human patients suffering from BOR syndrome with renal abnormalities, including hypoplasia and unilateral agenesis, and conductive hearing loss. In contrast, Eya1 homozygote mice die at birth, showing severe craniofacial and skeletal defects and the complete absence of the thymus, parathyroid glands, ears, and kidneys due to defective inductive tissue interactions (57, 59). Interestingly, similar to EYA1, the paralogous EYA4 gene has also been demonstrated to be associated with human disease. EYA4 is most closely related to EYA1 rather than EYA2 and EYA3. Mutations in EYA4 have been linked to cardiomyopathy and hearing loss (37, 42, 55). In contrast, there is no evidence for the existence of disease-associated mutations in EYA2 and EYA3 up to now. The identification and characterization of an Eya1 ortholog in zebrafish revealed a remarkable conservation in both the structure and expression pattern between Eya1 genes in fish and humans. In zebrafish, eya1 expression was detected in several organs during embryogenesis, such as the ears, the lateral line organ, the branchial arches, and the somites (41). As in mammals, eya1 in zebrafish is essential for the development of several tissues and organs. A quite well-studied example is the zebrafish ear, whose proper formation depends on eya1 expression, as shown by morpholino-mediated knockdown analysis (23). Since Eya1 is involved in the development of many different organs, we proposed that additional factors bind to it and, thereby, modulate its function. Using a yeast two-hybrid-based approach, we identified the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as so-far-unknown Eya1 interaction partners. We characterized the interactions and demonstrated that both Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Furthermore, we showed that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. Importantly, the morpholino-mediated knockdown of Sipl1 and Rbck1 orthologs in zebrafish led to a BOR syndrome-like phenotype, indicating the relevance of the Eya1-Sipl1/Rbck1 interaction in vivo.