Abstract

ABSTRACTSingle-nucleotide mutations in human SIX1 result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, and cause ∼4% of branchio-otic (BOS) and branchio-oto-renal (BOR) cases. The phenotypic variation between patients with the same mutation, even within affected members of the same family, make it difficult to functionally distinguish between the different SIX1 mutations. We made four of the BOS/BOR substitutions in the Xenopus Six1 protein (V17E, R110W, W122R, Y129C), which is 100% identical to human in both the protein-protein interaction domain and the homeodomain, and expressed them in embryos to determine whether they cause differential changes in early craniofacial gene expression, otic gene expression or otic morphology. We confirmed that, similar to the human mutants, all four mutant Xenopus Six1 proteins access the nucleus but are transcriptionally deficient. Analysis of craniofacial gene expression showed that each mutant causes specific, often different and highly variable disruptions in the size of the domains of neural border zone, neural crest and pre-placodal ectoderm genes. Each mutant also had differential effects on genes that pattern the otic vesicle. Assessment of the tadpole inner ear demonstrated that while the auditory and vestibular structures formed, the volume of the otic cartilaginous capsule, otoliths, lumen and a subset of the hair cell-containing sensory patches were reduced. This detailed description of the effects of BOS/BOR-associated SIX1 mutations in the embryo indicates that each causes subtle changes in gene expression in the embryonic ectoderm and otocyst, leading to inner ear morphological anomalies.

Highlights

  • Branchio-otic (BOS) and branchio-oto-renal (BOR) syndromes together comprise the second most prevalent birth defect involving hearing loss (Hilgert et al, 2009; Smith, 2018)

  • To exclude the possibility that the lack of transcriptional activation was caused by the mutations blocking Six1 from either localizing to the cell nucleus or translocating Eya1 to the nucleus, cells were co-transfected with FLAG-tagged versions of Six1WT or each Six1 mutant plus Myc-tagged Eya1 followed by confocal immunofluorescence microscopy (Fig. 1C-T)

  • This assay showed that R110W, W122R and Y129C efficiently translocate all of the transfected Eya1 into the nucleus; in no cell was Eya1 detected in the cytoplasm

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Summary

Introduction

Branchio-otic (BOS) and branchio-oto-renal (BOR) syndromes together comprise the second most prevalent birth defect involving hearing loss (Hilgert et al, 2009; Smith, 2018). These syndromes, which are inherited in an autosomal dominant manner with 100% penetrance (Chang et al, 2004; Fraser et al, 1980), are characterized. There is considerable variability in the severity, presence and type of these abnormalities among patients, including family members carrying the same mutation How these varied defects arise in the embryo has not yet been addressed

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