- Auditory defects and disorders are prevalent at all ages and affect 8% of the population in developed nations including newborns and children. Congenital hearing loss is the most common birth defect and it is estimated that 1 in 1000 children are affected by deafness at birth or before the onset of speech. Most of these children suffer from non-syndromic hearing loss, where hearing loss is the sole symptom with no other associated symptoms. More than two-thirds of non-syndromic hearing loss cases are of genetic origin, with approximately 80% inherited in an autosomal recessive mode. Autosomal recessive deafness is attributed to DFNB loci. Different DFNB loci have been reported and more than 50 genes have been identified to date. One such gene that encodes otoferlin, OTOF is responsible for the DFNB9 nonsyndromic form of deafness, which accounts for up to 8% of all prelingual autosomal recessive nonsyndromic deafness. Patients with mutations in
otoferlin suffer from profound sensorineural prelingual non-syndromic hearing loss.
Otoferlin belongs to the ferlin family of proteins. Ferlins are a group of multi-C2 domain proteins with emerging roles in vesicle fusion, membrane trafficking and repair. Otoferlin has six C2 domains including a C-terminal trans-membrane domain and these C2 domains bind to calcium, phospholipids and SNARE proteins. Otoferlin has been proposed to play a role in exocytosis of synaptic vesicles at the auditory inner hair cell synapse and also contributes to the development of the auditory cell synapse. Ablation of this gene causes abolition of exocytosis and improper targeting of synaptic components in sensory hair cells. More than 60 pathogenic mutations have been reported and are distributed in different C2, non-C2, and transmembrane domains of otoferlin. Despite the fact that otoferlin is a major contributor to sensorineural deafness, very little is known about the actual role of this protein in sensory hair cells.
Currently, the field is restricted by a lack of an in vivo model that could facilitate rapid analysis and easy characterization of otoferlin. Most studies have used mouse and rat models where, hair cells are few in number and require harvesting through time-consuming micro-dissections of the animal ear. Given the importance of otoferlin in hair cell development and function, and the lack of an easy genetically manipulatable model, the goal of this thesis was to develop a more tractable organismal model to establish the role of otoferlin.
Over the past years, zebrafish has emerged as an ideal model to study vertebrate development. It allows rapid assessment of gene function in vivo because it is amenable to genetic manipulation. Most importantly, the transparency of the zebrafish embryos allows direct visualization of tissue morphogenesis as it occurs in a live organism. Moreover, zebrafish possesss the easily accessible lateral line system, comprised of clusters of sensory hair cells, which are similar to mammalian type II vestibular hair cells and have similar morphological and anatomical structure. These sensory cells possesss synaptic ribbon bodies composed of L-type voltage activated calcium channels, a VGlut3 transporter for glutamate loading in synaptic vesicles, and develop a post-synapse upon maturation. Despite all these structural similarities zebrafish hair cells possesss only 3-5 synapses per hair cell. The aim here is to characterize the role of otoferlin by generating zebrafish models for otoferlinopathy.
Here we show for the first time that otoferlin is conserved in all species including zebrafish. However, due to a zebrafish genome duplication event, the otoferlin gene is paralogous, and there are two different subtypes which we named otoferlin a (encoded on chromosome 20) and otoferlin b (encoded on chromosome 17). Otoferlin a is the longer gene and has all six C2 domains, whereas otoferlin b is the shorter gene and lacks an N-terminal C2 domain. Otoferlin expression starts early during larval development and coincides with the onset of sensory morphogenesis and maturation. The otoferlin a isoform is strictly restricted to the hair cells of the developing otic
region, whereas otoferlin b is distributed in the hair cells of the otic region and lateral line neuromasts. At the cellular level, otoferlin distribution is observed in the supranuclear and basolateral regions of hair cells. Depleting both isoforms of otoferlin results in auditory and vestibular defects in larval zebrafish, and larvae exhibit abnormal swimming behavior. Moreover, larvae fail to develop an inflated swim bladder and develop a curved spine phenotype that becomes more prominent with development and facilitates a circling swimming behavior. These otoferlin-depleted zebrafish morphants belong to a class of ‘circler’ mutants that possesss similar hearing and balance defects. At the molecular level, sensory hair cells of otoferlin-depleted zebrafish developed atypical synapses comprised of tightly coalesced synaptic ribbon bodies, diffusely distributed VGlut3 and a dense and enlarged post-synapse. However, these hair cells possesssed an intact calcium response to mechanical deflection of hair bundles indicating an absence of mechanotransduction defects upon otoferlin depletion. Furthermore, otoferlin- depleted zebrafish exhibited severe defects in endocytotic dye uptake predominantly in the basolateral region of neuromast hair cells.
Studies have identified several otoferlin-interacting partners, however, a comprehensive study on otoferlin mutants is lacking. Also lacking is a more vivid description of the genes regulating the circler phenotype during development and maturation. For the first time we report a high-throughput transcriptomic analysis of otoferlin-depleted zebrafish in an attempt to
characterize the circler phenotype in larvae that also exhibit abnormal synaptic development. This study validates zebrafish as a model for high-throughput studies of the auditory and vestibular circler motility mutants, including otoferlin mutants. In this high-throughput transcriptomic study we identify several novel transcripts that are up or downregulated due to otoferlin depletion in zebrafish larvae. Some of these transcripts are very specific to the lateral line neuromasts and otic vesicle and have been shown to play roles during morphogenesis and development of the zebrafish sensory and neuronal regions. Moreover, there is a correlation between altered levels of some of these novel transcripts in otoferlin-depleted sensory cells and their synaptic morphology; hair cells with reduced levels of identified transcripts show an abnormal synaptic morphology indicated by coalesced distribution of synaptic ribbons when compared with control siblings.
Finally, this is also the first study that shows that the rescue of gross phenotypic loss due to otoferlin depletion is possible by co-injecting otoferlin-depleted larvae with the p5E-pmyo6b hair cell specific vector containing full length (FL) and truncated versions of mouse otoferlin constructs. This further reiterates that otoferlin is indeed conserved across species and that gross rescue of the phenotype can be achieved with either mouse otoferlin FL or truncated constructs that contains as little as just one C-terminal C2 domain with the trans-membrane region. This also provides a sense of which otoferlin domains are sufficient to rescue synaptic activity. Furthermore, we also see rescue in levels of reduced transcripts that are derived from the transcriptomic
analysis when otoferlin-depleted larvae are coinjected with the p5E-pmyo6b mouse FL vector construct.
Overall, these studies successfully establish zebrafish as an in vivo model organism to characterize defects associated with otoferlin loss, both at the molecular and transcriptomic levels in a much shorter span of time compared to mouse-based studies. They also show a promising application of otoferlin trans-species rescue using hair-cell-specific otoferlin rescue contructs that correct for loss of overall auditory and vestibular defects associated with otoferlin depletion in larval zebrafish.