Urinary tract malformations constitute the most frequent cause of chronic renal failure in the first two decades of life. Branchio-otic (BO) syndrome is an autosomal dominant developmental disorder characterized by hearing loss. In branchio-oto-renal (BOR) syndrome, malformations of the kidney or urinary tract are associated. Haploinsufficiency for the human gene EYA1, a homologue of the Drosophila gene eyes absent (eya), causes BOR and BO syndromes. We recently mapped a locus for BOR͞BO syndrome (BOS3) to human chromosome 14q23.1. Within the 33-megabase critical genetic interval, we located the SIX1, SIX4, and SIX6 genes, which act within a genetic network of EYA and PAX genes to regulate organogenesis. These genes, therefore, represented excellent candidate genes for BOS3. By direct sequencing of exons, we identified three different SIX1 mutations in four BOR͞BO kindreds, thus identifying SIX1 as a gene causing BOR and BO syndromes. To elucidate how these mutations cause disease, we analyzed the functional role of these SIX1 mutations with respect to proteinprotein and protein-DNA interactions. We demonstrate that all three mutations are crucial for Eya1-Six1 interaction, and the two mutations within the homeodomain region are essential for specific Six1-DNA binding. Identification of SIX1 mutations as causing BOR͞BO offers insights into the molecular basis of otic and renal developmental diseases in humans.

The murine Six gene family, homologous to Drosophila sine oculis(so) which encodes a homeodomain transcription factor, is composed of six members (Six1-6). Among the six members, only the Six2gene has been previously shown to be expressed early in kidney development,but its function is unknown. We have recently found that the Six1gene is also expressed in the kidney. In the developing kidney, Six1is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression became restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, we have recently generated Six1 mutant mice. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1-/- kidney development, we have found that Pax2, Six2 and Sall1expression was markedly reduced in the metanephric mesenchyme at E10.5,indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression was unaffected in Six1-/- metanephric mesenchyme at E10.5,indicating that Eya1 may function upstream of Six1. Moreover, our results show that both Eya1 and Six1expression in the metanephric mesenchyme is preserved in Pax2-/- embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, our results show that Eya1 and Six1genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development.

SUMMARY Inner ear hair cell differentiation requires Atoh1 function, while Eya1, Six1 and Sox2 are coexpressed in sensory progenitors and mutations in these genes cause sensorineural hearing loss. However, how these genes are linked functionally and the transcriptional networks controlling hair cell induction remain unclear. Here, we show that Eya1/Six1 are necessary for hair cell development and their coexpression in mouse cochlear explants is sufficient to induce hair cell fate in the nonsensory epithelium expressing low level Sox2 by activating not only Atoh1-dependent but also -independent pathways and that both pathways induce Pou4f3 to promote hair cell differentiation. Sox2 cooperates with Eya1/Six1 to synergistically activate Atoh1 transcription via direct binding to the conserved Sox- and Six-binding sites in Atoh1 enhancers and these proteins physically interact. Our findings demonstrate that direct and cooperative interactions between the Sox2, Six1 and Eya1 proteins coordinate Atoh1 expression to specify hair cell fate.