In this study, we used comparative genome sequencing and cytogenetics to examine the evolutionary history and the genomic context of three Otop genes in 25 evolutionarily diverse vertebrate species. We also extended our evolutionary studies to the Ush1g deafness gene because of the tight head-to-tail physical clustering of Ush1g with Otop2 and Otop3 in vertebrate genomes, and because mutations in Otop1 and Ush1g result in inner ear phenotypes in vertebrates. Based on our analyses, we conclude that the evolution of the Otop family in hominoids, amphibians, and rodents significantly departs from that of most vertebrate genomes, as does the evolution of Ush1g in the teleostei fish lineages.
The most striking difference found between hominoid species and other vertebrates is that the OTOP1 locus is flanked by a large SD of high complexity and sequence identity, belonging to the TBSD family, and arranged in an inverted orientation. Furthermore, OTOP1 sits only 3 kb away from the OTOP1 -proximal inversion boundary. Thus, the immediate genomic context of human OTOP1 differs significantly from that of mouse and zebrafish, the only other vertebrates in which Otop1 has been closely studied [4, 10]. Therefore, information on regulation of mouse and zebrafish Otop1 may not accurately reflect human OTOP1 regulation, and/or Otop1 developmental and biochemical function(s) in mouse and fish may be represented by another OTOP gene in humans. Using cytogenetic and comparative genomic approaches, we examined the evolutionary history of the hominoid Otop1 locus, including the flanking TBSD duplicons. Our findings indicate that the TBSD family emerged at some point after the divergence of the human, chimpanzee, and orangutan common ancestor from the macaque lineage ~12-16 million years ago, and later underwent significant expansion (perhaps within the common ancestor of the great apes). Further, these SDs contribute to plasticity and instability in multiple regions of the genome .On human chromosome 4p16 we describe a large (~5 Mb) inversion polymorphism flanked by palindromic TBSD sequences with OTOP1 as the boundary gene; this polymorphic arrangement, occurring in one in eight individuals of the Caucasian population, likely originated in the common human-chimpanzee lineage prior to ~6 million years ago.
Our studies, combined with others , have yielded a predicted timeline of genomic events affecting human chromosome loci 4p16 and 8p23 that have contributed to the structural complexity and genomic instability of the OTOP1 locus in humans; this timeline highlights interesting evolutionary parallels between these two regions. The macaque and mouse genomes are orthologous across the Otop1 locus, meaning that Otop1 and Drd5 are tightly linked, with no evidence for the presence of 7E OR gene clusters, complex SDs, or RS447 microsatellite sequences. RS447 sequences are, however, present in the macaque region orthologous to human chromosome 8p23. In orangutan, clusters of 7E OR and RS447 sequences reside close to each other in both regions, yet there is no evidence for complex mosaic duplications or inversions in either location. Chimpanzee (and bonobo) show increased copy number of the RS447 megasatellite on chromosome 4p16 (as deduced by array CGH; see additional file 3) and evidence for significant expansion of the TBSD family across a number of chromosomes. Furthermore, inversion polymorphisms of similar size (~5 Mb) have been reported on chromosomes 8p23 and 4p16 in humans and chimpanzees.
The mechanism underlying such genomic rearrangements is not completely clear. One possible scenario is that flanking clusters of SDs triggered the inversions via a non-allelic homologous recombination event (; and present study). Alternatively, rather than rearrangements being mediated by the duplications themselves, the inversions could have helped to create the complex segmental duplication architecture present at their breakpoints . Whatever the causal mechanism, the frequency of 8p23 and 4p16 inversion polymorphisms is relatively high in the human population, oftentimes with serious consequences for double heterozygotes. Specifically, mothers who are double heterozygotes for the inversion polymorphisms on chromosomes 4p16 and 8p23 are prone to recurrent de novo t(4;8)(p16;p23) translocations through unusual meiotic exchanges, resulting in offspring with Wolf-Hirschhorn syndrome. The phenotype of patients with this syndrome consists of mental retardation and an array of developmental defects, often including hypotonia (decreased muscle tone; [15, 44, 45]). It is conceivable that individuals broadly labeled hypotonic might also suffer from vestibular dysfunction, which could go undiagnosed because it is often not clinically assessed. It would thus be interesting to study Wolf-Hirschhorn syndrome patients with balanced t(4;8)(p16;p23) translocations in search of individuals with disrupted or dysregulated OTOP1 (via OTOP1 copy number changes, formation of OTOP1 chimeras, or altered OTOP1 position). The resulting physiological consequences could include complete abrogation of OTOP1 function, haploinsufficiency, elevated OTOP1 expression, or creation of a fusion protein with a dominant-negative effect or novel gain of function , any of which would contribute to our understanding of the role of OTOP1 in human development.
X. tropicalis is the other vertebrate in which we found significant differences in genomic organization of the Otop family. Specifically, the X. tropicalis genome contains multiple paralogs for each Otop gene. We have determined that amphibian Nlo has a phylogenetic relationship to Otop3 -like genes. Both Nlo and Otop1 are apparently involved in the development of the vestibular system in amphibians and placental mammals, respectively, suggesting that multiple Otop genes may have roles in vestibular system development. Studying the degree of sub-functionalization of Otop paralogs in X. tropicalis may help to define the unique functions attributable to each paralog.
Amphibians and other vertebrates share similar auditory and vestibular physiology, with their vestibular organs particularly well conserved in position, structure, and function . However, a striking difference is the unusual calcium carbonate crystalline forms of amphibian otoconia. The general trend during vertebrate evolution has been a replacement of otoconial vaterite and aragonite crystals by calcite, a calcium carbonate crystal polymorph of increased stability . Such a trend correlates with the transition of vertebrates from aquatic to terrestrial life. Chondrostei fish have vaterite and/or aragonite crystals, teleostean fish only have aragonitic otoliths, and both mammalian and avian otoconia consist exclusively of calcite crystals. During vertebrate evolution, calcitic otoconia appeared for the first time in the amphibian inner ear, yielding an intriguing intermediate situation with two crystalline forms: calcite in the utricle and aragonite in the saccule, lagena, and endolymphatic sac [49–52].
The structural variation of otoconia among vertebrates is thought to result from different properties of their scaffold proteins. However, the main scaffold protein in amphibians, otoconin-22 (Oc-22), is produced in most portions of the developing inner ear (i.e., saccule, crista ampularis, endolymphatic sac, and utricule ), making it unclear how two entirely different crystalline forms of otoconia are regionally specified during amphibian development. Therefore, additional factors unique to either the utricule (with calcitic otoconia) or saccule (with aragonitic otoconia) likely lead to distinct regional nucleation and crystal growth during otoconial formation in amphibians. Reverse genetic screens that might clarify the peculiarities of otoconial formation in amphibians have been impossible to undertake because animal models with clean, non-syndromic balance phenotypes are generally very rare , to the point that frog mutants with complete otoconial agenesis have yet to be described. In this regard, a candidate gene approach involving targeted disruption of Otop genes in X. tropicalis, individually or in combinations, could help discern whether different Otop paralogs have acquired discrete functions contributing to the regional differences in otoconial formation; such a study could be performed using morpholinos, which have been successfully used to specifically examine the role of Otop1 in vestibular system development in zebrafish  and general otic vesicle development in Xenopus .
Of the 16,000 deaf-blind persons in the United States, more than half are believed to have Usher syndrome (USH), a combination of progressive retinopathy and congenital hearing loss. USH type I is especially devastating because of its early onset and extreme phenotype of profound congenital deafness with unintelligible speech, retinitis pigmentosa within the first decade of life, and vestibular dysfunction . USHIG is one of five USH type 1 causative genes identified to date. Those include the genes encoding the molecular motor myosin VIIa (MYO7A; USH1B); two cell adhesion cadherin proteins, cadherin 23 (CDH23; USH1D) and protocadherin 15 (PCDH15; USH1F); and two scaffold proteins, harmonin (harmonin; USH1C) and USH1G (USH1G; USH1G). These proteins are involved in a protein network or "interactome" within sensory cells of the eye and inner ear .
Our evolutionary studies of Ush1g in vertebrates revealed a unique expansion of this gene family to include three members in all five fish species studied. A number of zebrafish models of USH have been described, including mariner (Myo7A, ), sputnik (Cdh23, ), and orbiter (Pcdh15a, ). Our efforts have revealed that multiple copies of Myo7A, Cdh23, Pcdh15, and Ush1g are present in fish genomes (two, two, two, and three copies, respectively; data not shown). Interestingly, there is evidence that the "USH interactome" may be more complex in fish than in other vertebrates . Specifically, the two Pcdh15 orthologs in zebrafish have distinct functions in hearing and vision: Pcdh15a is required for normal auditory and vestibular function, while Pcdh15b is required for normal photoreceptor outer segment organization and retinal function. Because Ush1g is a scaffold protein, the presence of three Ush1g genes in fish may affect the fish "USH interactome" (and consequently eye and inner-ear development) differently than in humans; such differences should be considered when using fish systems as general models for vertebrate ear physiology.
Functional studies that shed light on the cis-regulatory elements and fine genomic architecture of the Ush1g - Otop2 locus are needed to establish the mechanisms underlying the lineage-specific differences in Ush1g function. For instance, like human USH1g patients, the Ush1g -defective mouse model Js is profoundly deaf with vestibular dysfunction; however, unlike human USH1g patients, it does not have abnormal retinal phenotype . It is notable that the transcriptional boundaries between Ush1g and Otop2 are indistinct in rodent genomes due the presence of two rodent-specific Otop2, 5' untranslated exons that cause Ush1g to overlap with (or be embedded within) the Otop2 transcriptional unit; the effect of this configuration on the expression of these two genes is not known.
CTCF is a zinc-finger transcriptional repressor that serves an insulator function to limit the spread of heterochromatin; it can also operate as a transcriptional activator, regulate nuclear localization, and participate in the control of imprinting . The first intron of Ush1g contains an extensive number of evolutionarily conserved sequences and is bracketed by two putative CTCF-binding sites, CTCF2 and CTCF3. As a caveat, the predicted CTCF binding sites are based on positional information of sequence reads in chIP-seq studies and need to be further validated. Nonetheless, we suggest that the HighOc CTCF3 site between Ush1g and Otop2 may be ubiquitously bound by CTCF in mouse and human, while CTCF1, CTCF2, and CTCF4 may participate in gene expression regulation in a cell- and/or species-specific fashion. Further functional characterization of CTCF1 to CTCF4 will be pivotal for determining whether Ush1g and Otop are functionally insulated or participate in common developmental pathways.