Sobp is a novel Six1 co‐factor during inner ear development

Abstract

According to the CDC, the prevalence of hearing loss is 1.4 per 1000 babies screened in 2009 in the USA. Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by hearing loss, branchial fistulas/cysts, and renal anomalies in about a third of patients. Mutations in the SIX1 gene and the SIX1 co-factor EYA1 gene are present in half of BOR patients. We are using Xenopus laevis and mouse as models to discover novel genes involved in this syndrome. Based on a screen in fly of proteins shown to interact with So (Drosophila Six1 homologue), sobp was identified as potential Six1 co-factor. Expression data show that this gene is expressed in patterns that overlap with six1 during craniofacial development. Co-IP and immunofluorescence experiments demonstrate that Sobp binds to and co-localizes with Six1 in the cell nucleus. Additionally, Six1 is able to bind and translocate to the cell nucleus an Sobp construct lacking its nuclear localization signal further demonstrating interaction. Luciferase assays to test if this factor is able to modify Six1 function show that Sobp represses Six1+Eya1 transcriptional activity. Knockdown experiments using morpholino antisense oligonucleotides and CRISPR/Cas9 followed by ISH and qPCR analyses of embryos at late neural plate stages show that Sobp is required for neural crest (foxd3) and placode (six1) formation. These changes are accompanied by an expansion of the neural plate (sox2) and reduction of the embryonic epidermis (krt12.4). Sobp overexpression confirmed that Sobp is required for neural crest (foxd3) and placode (six1) formation; changes in those two genes were accompanied by a slight expansion of the krt12.4-expressing domain. With further development, Sobp knockdown and overexpression disrupt otocyst development shown by disrupted expression of dlx5, pax2, irx1 and sox9 at tailbud stages, and cause severe craniofacial defects in the tadpole. These results indicate that Sobp is required for proper craniofacial development, and that it modifies Six1 function, suggesting this protein may be a potential candidate gene for BOR and other craniofacial defects.