Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Abstract

Sensory hair cells convert mechanical motion into chemical signals.Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance.H air cells couple mechanical motion to neurotransmitter release at synapses (1). In contrast to conventional neural synapses, hair cell synapses release neurotransmitter continuously and, in a graded manner (2), possess synaptic ribbons (2-4), and lack synaptophysin (5), complexin (6-9), Munc13 (10), and the calcium sensors synaptotagmin I and II (11). In place of synaptotagmin, it is believed that otoferlin may confer calcium sensitivity to evoke neurotransmitter release (12, 13). Otoferlin is a six-C2 domain transmembrane protein expressed in inner, outer, and vestibular hair cells, as well as restricted regions of the brain (13-16). In humans, missense mutations in otoferlin have been linked to hearing loss (17,18), and biochemical studies have determined that otoferlin binds calcium and lipids (12,19), as well as membrane trafficking proteins (12,(20)(21)(22)(23). Further, in vitro assays have demonstrated that otoferlin accelerates SNARE-mediated membrane fusion (12). Based upon this evidence, it is hypothesized that otoferlin functions as a calcium-sensitive regulator of neurotransmitter release in sensory hair cells.However, the results of several studies have raised questions related to otoferlin's function. For instance, despite otoferlin expression in vestibular hair cells, knockout mice show no balance defects (24, 25) despite reduced exocytosis in vestibular type I hair cells (24,26). This raises questions as to the importance of otoferlin in this system. Further, otoferlin did not rescue synchronous neurotransmitter release in synaptotagmin I knockout cultured neurons, indicating that otoferlin and synaptotagmin are not functionally redundant (2...

Transcriptomic Analyses of Inner Ear Sensory Epithelia in Zebrafish

“…Figure 11 shows the expression of over 80 genes that have been implicated in human deafness or related to hearing in animal models. col11a1a, col11ab, col11a2, col4a5, dfna5b, eya1, eya2, grhl2a, grhl2b, kcnq4, otofa, otofb, and slc26a5 are highly expressed in the zebrafish otic vesicle, hair cells, and semicircular canal (Croushore , 2005;Blasiole et al, 2006;Albert et al, 2007;Xiao and Baier, 2007;Baas et al, 2009;Yokoi et al, 2009;Fang et al, 2010;Hoffman et al, 2010;Janicke et al, 2010;Landgraf et al, 2010;Wu et al, 2014;Chatterjee et al, 2015;Wang et al, 2015). col11a1 has been shown to express in the inner ear and otic capsule of mice (Yoshioka et al, 1995;Diez-Roux et al, 2011) and spontaneous mutation mouse models show both auditory and vestibular abnormalities.…”

Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

“…Our observation that multiple C2 domains interact with Cav1.3 suggests that some C2 domains may be somewhat functionally redundant. Indeed, it was reported that exogenous truncated forms of otoferlin lacking one or more C2 domains could rescue balance and startle reflex in zebrafish lacking endogenous otoferlin (25). This observation may be explained if the truncated otoferlin constructs retained at least one C2 domain capable of binding Cav1.…”

Ribbon synapses in zebrafish hair cells

“…Otoferlin has been detected in hair cells of multiple species including zebrafish (Goodyear et al, 2010). A recent study using morpholinos against otoferlin a and otoferlin b showed that double knockdown resulted in defective balance and hearing in zebrafish larvae (Chatterjee et al, 2015). Rescue experiments using various deletions of the C2 domains revealed that the C-terminal C2 domains are critical for function (Chatterjee et al, 2015).…”

“…A recent study using morpholinos against otoferlin a and otoferlin b showed that double knockdown resulted in defective balance and hearing in zebrafish larvae (Chatterjee et al, 2015). Rescue experiments using various deletions of the C2 domains revealed that the C-terminal C2 domains are critical for function (Chatterjee et al, 2015). Aside from a severe reduction of exocytosis in hair cells, which is presumably the case in the zebrafish knockdowns, the deletion of otoferlin in mice results in a significant decrease in the number of ribbons (Roux et al, 2006).…”