Sobp modulates the transcriptional activation of Six1 target genes and is required during craniofacial development.

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR.

A different type of branchial fistula as part of a branchiootorenal syndrome.

We describe an extremely rare case of a complete fistula, a combination of the first 2 branchial arches as a component of branchiootorenal syndrome. A 13-year-old girl presented with the complaint of intermittent drainage from bilateral preauricular and right lower neck external openings. A contrast fistulogram revealed a complete fistula. Diagnostic features and surgical techniques are discussed in detail.

SIX1 acts synergistically with TBX18 in mediating ureteral smooth muscle formation.

Dysfunction of the ureter often leads to urine flow impairment from the kidney to the bladder, causing dilation of the ureter and/or renal pelvis. Six1 is a crucial regulator of renal development: mutations in human SIX1 cause branchio-oto-renal (BOR) syndrome and Six1(-/-) mice exhibit renal agenesis, although the ureter is present. It remains unclear whether Six1 plays a role in regulating ureter morphogenesis. We demonstrate here that Six1 is differentially expressed during ureter morphogenesis. It was expressed in undifferentiated smooth muscle (SM) progenitors, but was downregulated in differentiating SM cells (SMCs) and had disappeared by E18.5. In Six1(-/-) mice, the ureteral mesenchymal precursors failed to condense and differentiate into normal SMCs and showed increased cell death, indicating that Six1 is required for the maintenance and normal differentiation of SM progenitors. A delay in SMC differentiation was observed in Six1(-/-) ureters. A lack of Six1 in the ureter led to hydroureter and hydronephrosis without anatomical obstruction when kidney formation was rescued in Six1(-/-) embryos by specifically expressing Six1 in the metanephric mesenchyme, but not the ureter, under control of the Eya1 promoter. We show that Six1 and Tbx18 genetically interact to synergistically regulate SMC development and ureter function and that their gene products form a complex in cultured cells and in the developing ureter. Two missense mutations in SIX1 from BOR patients reduced or abolished SIX1-TBX18 complex formation. These findings uncover an essential role for Six1 in establishing a functionally normal ureter and provide new insights into the molecular basis of urinary tract malformations in BOR patients.

Catweasel mice: a novel role for Six1 in sensory patch development and a model for branchio-oto-renal syndrome.

Large-scale mouse mutagenesis initiatives have provided new mouse mutants that are useful models of human deafness and vestibular dysfunction. Catweasel is a novel N-ethyl-N-nitrosourea (ENU)-induced mutation. Heterozygous catweasel mutant mice exhibit mild headtossing associated with a posterior crista defect. We mapped the catweasel mutation to a critical region of 13 Mb on chromosome 12 containing the Six1, -4 and -6 genes. We identified a basepair substitution in exon 1 of the Six1 gene that changes a conserved glutamic acid (E) at position 121 to a glycine (G) in the Six1 homeodomain. Cwe/Cwe animals lack Preyer and righting reflexes, display severe headshaking and have severely truncated cochlea and semicircular canals. Cwe/Cwe animals had very few hair cells in the utricle, but their ampullae and cochlea were devoid of any hair cells. Bmp4, Jag1 and Sox2 expression were largely absent at early stages of sensory development and NeuroD expression was reduced in the developing vestibulo-acoustic ganglion. Lastly we show that Six1 genetically interacts with Jag1. We propose that the catweasel phenotype is due to a hypomorphic mutation in Six1 and that catweasel mice are a suitable model for branchio-oto-renal syndrome. In addition Six1 has a pivotal role in early sensory patch development and may act in the same genetic pathway as Jag1.

A novel nonsense mutation in the EYA1 gene associated with branchio-oto-renal/branchiootic syndrome in an Afrikaner kindred.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by the associations of hearing loss, branchial arch defects and renal anomalies. Branchiootic (BO) syndrome is a related disorder that presents without the highly variable characteristic renal anomalies of BOR syndrome. Dominant mutations in the human homologue of the Drosophila eyes absent gene (EYA1) are frequently the cause of both BOR and BO syndromes. We report a South African family of Afrikaner descent with affected individuals presenting with pre-auricular abnormalities and either hearing loss or bilateral absence of the kidneys. Genetic analysis of the pedigree detected a novel EYA1 heterozygous nonsense mutation in affected family members but not in unaffected family members or a random DNA panel. Through mutational analysis, we conclude that this particular mutation is the cause of BOR/BO syndrome in this family as a result of a truncation of the EYA1 protein that ablates the critical EYA homologous region. To the best of our knowledge, this is the first case of BOR/BO syndrome reported in Africa or in those of the Afrikaner descent.

Eya1 expression in the developing ear and kidney: towards the understanding of the pathogenesis of Branchio-Oto-Renal (BOR) syndrome.

Branchio-Oto-Renal (BOR) syndrome is an autosomal dominant, early developmental defect characterised by varying combinations of branchial (fistulas, sinuses, and cysts), outer, middle and inner ear, and renal anomalies. The gene underlying this syndrome, EYA1, is homologous to the Drosophila developmental gene eyes absent which encodes a transcriptional co-activator required for eye specification. We report here the temporal and spatial pattern of expression of the murine homologue, Eya1, throughout ear and kidney development in relation to the anomalies of BOR syndrome. The expression of Eya1 in the branchial arch apparatus (namely in the 2nd, 3rd, and 4th branchial clefts and pharyngeal pouches) at embryonic day (E)10.5, can be correlated with the branchial fistulas, sinuses, and cysts but not with the outer and middle ear anomalies. In contrast, Eya1 is expressed during the slightly more advanced stage of outer and middle ear morphogenesis at E13.5, in the mesenchyme adjacent to the first branchial cleft (the cleft will give rise to the external auditory canal and the surrounding mesenchyme to the auricular hillocks) and surrounding the primordia of the middle ear ossicles, and in the epithelium of the tubotympanic recess (the future tympanic cavity). During early inner ear development, Eya1 is expressed in the ventromedial wall of the otic vesicle (the site of the future sensory epithelia), in the statoacoustic ganglion, and in the periotic mesenchyme, consistent with the cochlear anomalies and sensorineural hearing loss of BOR syndrome. Subsequently, Eya1 expression is observed in the differentiating hair and supporting cells of the sensory epithelia, as well as in the associated ganglia, and persists after differentiation has taken place. This suggests that, in addition to a role in the morphogenetic process, Eya1 could also be implicated in the differentiation and/or survival of these inner ear cell populations. Finally, Eya1 expression in the condensing mesenchymal cells of the kidney is consistent with the excretory and collecting system anomalies of BOR syndrome. From the comparison of the Eya1 and Pax2 expression patterns during ear and kidney development, a contribution of these two genes to the same regulatory pathway can only be suggested in the mesenchymal-epithelial transition directing renal tubule formation.