Background: The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS.

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 branchiooto-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.

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, craniofacial and/or renal defects. Variants in the transcription factor Six1 and its cofactor Eya1, both required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 cofactor 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-IP and immunofluorescence experiments demonstrate that Sobp binds to and co-localizes 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 knockdown or increase expression show that Sobp 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, is required for vertebrate craniofacial development, and identifies Sobp as a potential candidate gene for BOR.

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