RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.     

Analysis of mouse kreisler mutants reveals new roles of hindbrain-derived signals in the establishment of the otic neurogenic domain

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3−/−Fgf10−/− double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis.     

Expression of proneural and neurogenic genes in the zebrafish lateral line primordium correlates with selection of hair cell fate in neuromasts

Expression of a mouse atonal homologue, math1, defines cells with the potential to become sensory hair cells in the mouse inner ear (Science 284 (1999) 1837) and Notch signaling limits the number of cells that are permitted to adopt this fate (Nat. Genet. 21 (1999) 289; J. Neurocytol. 28 (1999) 809). Failure of lateral inhibition mediated by Notch signaling is associated with an overproduction of ear hair cells in the zebrafish mind bomb (mib) and deltaA mutants (Development 125 (1998a) 4637; Development 126 (1999) 5669), suggesting a similar role for these genes in limiting the number of hair cells in the zebrafish ear. This study extends the analysis of proneural and neurogenic gene expression to the lateral line system, which detects movement via clusters of related sensory hair cells in specialized structures called neuromasts. We have compared the expression of a zebrafish atonal homologue, zath1, and neurogenic genes, deltaA, deltaB and notch3, in neuromasts and the posterior lateral line primordium (PLLP) of wild-type and mib mutant embryos. We describe progressive restriction of proneural and neurogenic gene expression in the migrating PLLP that appears to correlate with selection of hair cell fate in maturing neuromasts. In mib mutants there is a failure to restrict expression of zath1 and Delta homologues in the neuromasts revealing similarities with the phenotype previously described in the ear.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Cadherin expression in the inner ear of developing zebrafish

Cadherins are cell adhesion molecules that have been implicated in development of a variety of organs including the ear. In this study we analyzed expression patterns of three zebrafish cadherins (Cadherin-2, -4, and -11) in the embryonic and larval zebrafish inner ear using both in situ hybridization and immunocytochemical methods. All three Cadherins exhibit distinct spatiotemporal patterns of expression during otic vesicle morphogenesis. Cadherin-2 and Cadherin-4 proteins and their respective mRNAs were detected mainly in the sensory patches and the statoacoustic ganglion (SAg), respectively. In contrast, cadherin-11mRNA was widely expressed earlier in the otic placode, and later became restricted to a subset of cells in the inner ear, including hair cells.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

Expression of zebrafish hip: Response to Hedgehog signalling,comparison with ptc1 expression,and possible role in otic patterning

In zebrafish, Hedgehog (Hh) signalling is required to specify posterior otic identity. This presents a conundrum, as the nearest source of Hh to the developing inner ear is the ventral midline, in the notochord and floorplate. How can a source of Hh that is ostensibly constant with respect to the anteroposterior axis of the otic vesicle specify posterior otic identity? One possibility is that localised inhibition of Hh signalling is involved. Here we show that genes coding for three inhibitors of Hh signalling, su(fu), dzip1 and hip, are expressed in and around the developing otic vesicle. su(fu) and dzip1 are ubiquitously expressed and unaffected by Hh levels. The expression of hip, however, is positively regulated by Hh signalling and has a complex, dynamic pattern. It is detectable in the neural tube, otic vesicle, statoacoustic ganglion, brain, fin buds, mouth, somites, pronephros and branchial arches. These expression domains bear some similarity, but are not identical, to those of ptc1, a Hh receptor gene that is also positively regulated by Hh signalling. In the neural tube, for instance, hip is expressed in a subset of the ptc1 expression domain, while in other regions, including the otic vesicle, hip and ptc1 expression domains differ. Significantly, we find that initial expression of hip is higher in and adjacent to anterior otic regions, while ptc1 expression becomes progressively restricted to the posterior of the ear. Hip-mediated inhibition of Hh signalling may therefore be important in restricting the effects of Hh to posterior regions of the developing inner ear.     

Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development

Background

The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.

Methodology/Principal Findings

We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.

Conclusions/Significance

Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.     

The developing lamprey ear closely resembles the zebrafish otic vesicle: otx1 expression can account for all major patterning differences

The inner ear of adult agnathan vertebrates is relatively symmetric about the anteroposterior axis, with only two semicircular canals and a single sensory macula. This contrasts with the highly asymmetric gnathostome arrangement of three canals and several separate maculae. Symmetric ears can be obtained experimentally in gnathostomes in several ways, including by manipulation of zebrafish Hedgehog signalling, and it has been suggested that these phenotypes might represent an atavistic condition. We have found, however, that the symmetry of the adult lamprey inner ear is not reflected in its early development; the lamprey otic vesicle is highly asymmetric about the anteroposterior axis, both morphologically and molecularly, and bears a striking resemblance to the zebrafish otic vesicle. The single sensory macula originates as two foci of hair cells, and later shows regions of homology to the zebrafish utricular and saccular maculae. It is likely, therefore, that the last common ancestor of lampreys and gnathostomes already had well-defined otic anteroposterior asymmetries. Both lamprey and zebrafish otic vesicles express a target of Hedgehog signalling, patched, indicating that both are responsive to Hedgehog signalling. One significant distinction between agnathans and gnathostomes, however, is the acquisition of otic Otx1 expression in the gnathostome lineage. We show that Otx1 knockdown in zebrafish, as in Otx1(-/-) mice, gives rise to lamprey-like inner ears. The role of Otx1 in the gnathostome ear is therefore highly conserved; otic Otx1 expression is likely to account not only for the gain of a third semicircular canal and crista in gnathostomes, but also for the separation of the zones of the single macula into distinct regions.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear

Sox2 has been variously implicated in maintenance of pluripotent stem cells or, alternatively, early stages of cell differentiation, depending on context. In the developing inner ear, Sox2 initially marks all cells in the nascent sensory epithelium and, in mouse, is required for sensory epithelium formation. Sox2 is eventually downregulated in hair cells but is maintained in support cells, the functional significance of which is unknown. Here we describe regulation and function of sox2 in the zebrafish inner ear. Expression of sox2 begins after the onset of sensory epithelium development and is regulated by Atoh1a/b, Fgf and Notch. Knockdown of sox2 does not prevent hair cell production, but the rate of accumulation is reduced due to sporadic death of differentiated hair cells. We next tested the capacity for hair cell regeneration following laser ablation of mature brn3c:gfp-labeled hair cells. In control embryos, regeneration of lost hair cells begins by 12 h post-ablation and involves transdifferentiation of support cells rather than asymmetric cell division. In contrast, regeneration does not occur in sox2-depleted embryos. These data show that zebrafish sox2 is required for hair cell survival, as well as for transdifferentiation of support cells into hair cells during regeneration.     

Partially redundant proneural function reveals the importance of timing during zebrafish olfactory neurogenesis

Little is known about proneural gene function during olfactory neurogenesis in zebrafish. Here, we show that the zebrafish Atonal genes neurogenin1 (neurog1) and neurod4 are redundantly required for development of both early-born olfactory neurons (EONs) and later-born olfactory sensory neurons (OSNs). We show that neurod4 expression is initially absent in neurog1 mutant embryos but recovers and is sufficient for the delayed development of OSN. By contrast, EON numbers are significantly reduced in neurog1 mutant embryos despite the recovery of neurod4 expression. Our results suggest that a shortened time window for EON development causes this reduction; the last S-phase of EON is delayed in neurog1 mutant embryos but mutant EONs are all post-mitotic at the same stage as EONs in wild-type embryos. Finally, we show that expression of certain genes, such as robo2, is never detected in neurog1 mutant EONs. Failure of robo2 expression to recover correlates with defects in the fasciculation of neurog1 mutant olfactory axonal projections and in the organisation of proto-glomeruli because projections arrive at the olfactory bulb that are reminiscent of those in robo2 mutant embryos. We conclude that the duration of proneural expression in EON progenitors is crucial for correct development of the zebrafish olfactory system.     

An expanded domain of fgf3 expression in the hindbrain of zebrafish valentino mutants results in mis-patterning of the otic vesicle

The valentino (val) mutation in zebrafish perturbs hindbrain patterning and, as a secondary consequence, also alters development of the inner ear. We have examined the relationship between these defects and expression of fgf3 and fgf8 in the hindbrain. The otic vesicle in val/val mutants is smaller than normal, yet produces nearly twice the normal number of hair cells, and some hair cells are produced ectopically between the anterior and posterior maculae. Anterior markers pax5 and nkx5.1 are expressed in expanded domains that include the entire otic epithelium juxtaposed to the hindbrain, and the posterior marker zp23 is not expressed. In the mutant hindbrain, expression of fgf8 is normal, whereas the domain of fgf3 expression expands to include rhombomere 4 through rhombomere X (an aberrant segment that forms in lieu of rhombomeres 5 and 6). Depletion of fgf3 by injection of antisense morpholino (fgf3-MO) suppresses the ear patterning defects in val/val embryos: Excess and ectopic hair cells are eliminated, expression of anterior otic markers is reduced or ablated, and zp23 is expressed throughout the medial wall of the otic vesicle. By contrast, disruption of fgf8 does not suppress the val/val phenotype but instead interacts additively, indicating that these genes affect distinct developmental pathways. Thus, the inner ear defects observed in val/val mutants appear to result from ectopic expression of fgf3 in the hindbrain. These data also indicate that val normally represses fgf3 expression in r5 and r6, an interpretation further supported by the effects of misexpressing val in wild-type embryos. This is in sharp contrast to the mouse, in which fgf3 is normally expressed in r5 and r6 because of positive regulation by kreisler, the mouse ortholog of val. Implications for co-evolution of the hindbrain and inner ear are discussed.     

Sox2 and Fgf interact with Atoh1 to promote sensory competence throughout the zebrafish inner ear

Atoh1 is required for differentiation of sensory hair cells in the vertebrate inner ear. Moreover, misexpression of Atoh1 is sufficient to establish ectopic sensory epithelia, making Atoh1 a good candidate for gene therapy to restore hearing. However, competence to form sensory epithelia appears to be limited to discrete regions of the inner ear. To better understand the developmental factors influencing sensory-competence, we examined the effects of misexpressing atoh1a in zebrafish embryos under various developmental conditions. Activation of a heat shock-inducible transgene, hs:atoh1a, resulted in ectopic expression of early markers of sensory development within 2 h, and mature hair cells marked by brn3c:GFP began to accumulate 9 h after heat shock. The ability of atoh1a to induce ectopic sensory epithelia was maximal when activated during placodal or early otic vesicle stages but declined rapidly thereafter. At no stage was atoh1a sufficient to induce sensory development in dorsal or lateral non-sensory regions of the otic vesicle. However, co-misexpression of atoh1a with fgf3, fgf8 or sox2, genes normally acting in the same gene network with atoh1a, stimulated sensory development in all regions of the otic vesicle. Thus, expression of fgf3, fgf8 or sox2 strongly enhances competence to respond to Atoh1.