Six1 proteins with human branchio-oto-renal mutations differentially affect cranial gene expression and otic development

摘要

Single-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. 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 by deformities of the external, middle and inner ear structures, as well as second branchial arch defects that include fistulas and cysts; renal malformations also characterize BOR ( Kochhar et al., 2007 ; Smith, 2018 ). External ear malformations include pre-auricular pits, tags and cupped/lop ears; those of the middle ear include ossicle deformities, and of the inner ear include auditory and vestibular canal hypoplasia ( Smith, 2018 ). 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. Mutations in two genes have been identified in about half of patients with BOS/BOR (reviewed in Smith, 2018 ; Moody et al., 2015 ). The gene encoding the SIX1 transcription factor is mutated in ～4% of patients (BOS3/BOR3), and the gene encoding EYA1, which binds to SIX1 to modify its transcriptional activity, is mutated in ～40% of the patients (BOS1/BOR1). SIX1 is highly related to Drosophila Sine oculis (SO) ( Cheyette et al., 1994 ; Serikaku and O'Tousa, 1994 ; Kawakami et al., 1996 ). Like other proteins of the SIX family, SIX1 possesses two highly conserved domains: a SIX-type homeodomain (HD) that binds to DNA and an N-terminal SIX domain (SD) that binds to cofactor proteins including EYA1 ( Pignoni et al., 1997 ; Kawakami et al., 2000 ; Kobayashi et al., 2001 ; Kenyon et al., 2005a ). Several SIX1 single-nucleotide mutations that result mostly in amino acid substitutions have been reported in BOS/BOR patients ( Fig.?1 A), but only a few have been functionally analyzed and only in cell culture. The V17E mutation in the first α-helix of the SD is reported to abolish the SIX1-EYA interaction and subsequent translocation of EYA to the nucleus ( Patrick et al., 2009 ). The R110W mutation in the sixth α-helix of the SD is reported to decrease the SIX1-EYA interaction ( Kochhar et al., 2008 ; Ruf et al., 2004 ) and be deficient in transcriptional activation ( Patrick et al., 2009 ). The W122R mutation, adjacent to the HD ( Sanggaard et al., 2007 ), is predicted to affect DNA binding ( Patrick et al., 2009 ), and the Y129C mutation in the HD results in significantly reduced DNA binding ( Ruf et al., 2004 ; Patrick et al., 2009 ). To our knowledge, the gene expression consequences of these mutations on craniofacial development are unknown.