The role of jab1, a putative downstream effector of the neurotrophic cytokine macrophage migration inhibitory factor (MIF) in zebrafish inner ear hair cell development.

Macrophage migration inhibitory factor (MIF) is a neurotrophic cytokine essential for inner ear hair cell (HC) development and statoacoustic ganglion (SAG) neurite outgrowth, and SAG survival in mouse, chick and zebrafish. Another neurotrophic cytokine, Monocyte chemoattractant protein 1 (MCP1) is known to synergize with MIF; but MCP1 alone is insufficient to support mouse/chick SAG neurite outgrowth or neuronal survival. Because of the relatively short time over which the zebrafish inner ear develops (~30hpf), the living zebrafish embryo is an ideal system to examine mif and mcp1 cytokine pathways and interactions. We used a novel technique: direct delivery of antisense oligonucleotide morpholinos (MOs) into the embryonic zebrafish otocyst to discover downstream effectors of mif as well as to clarify the relationship between mif and mcp1 in inner ear development. MOs for mif, mcp1 and the presumptive mif and mcp1 effector, c-Jun activation domain-binding protein-1 (jab1), were injected and then electroporated into the zebrafish otocyst 25-48hours post fertilization (hpf). We found that although mif is important at early stages (before 30hpf) for auditory macular HC development, jab1 is more critical for vestibular macular HC development before 30hpf. After 30hpf, mcp1 becomes important for HC development in both maculae.

Ontogenetic development of the inner ear saccule and utricle in the Lusitanian toadfish: Potential implications for auditory sensitivity.

Studies addressing structure-function relationships of the fish auditory system during development are sparse compared to other taxa. The Batrachoididae has become an important group to investigate mechanisms of auditory plasticity and evolution of auditory-vocal systems. A recent study reported ontogenetic improvements in the inner ear saccule sensitivity of the Lusitanian toadfish, Halobatrachus didactylus, but whether this results from changes in the sensory morphology remains unknown. We investigated how the macula and organization of auditory receptors in the saccule and utricle change during growth in this species. Inner ear sensory epithelia were removed from the end organs of previously PFA-fixed specimens, from non-vocal posthatch fry (<1.4 cm, standard length) to adults (>23 cm). Epithelia were phalloidin-stained and analysed for area, shape, number and orientation patterns of hair cells (HC), and number and size of saccular supporting cells (SC). Saccular macula area expanded 41x in total, and significantly more (relative to body length) among vocal juveniles (2.3-2.9 cm). Saccular HC number increased 25x but HC density decreased, suggesting that HC addition is slower relative to epithelial growth. While SC density decreased, SC apical area increased, contributing to the epithelial expansion. The utricule revealed increased HC density (striolar region) and less epithelial expansion (5x) with growth, contrasting with the saccule that may have a different developmental pattern due to its larger size and main auditory functions. Both macula shape and HC orientation patterns were already established in the posthatch fry and retained throughout growth in both end organs. We suggest that previously reported ontogenetic improvements in saccular sensitivity might be associated with changes in HC number (not density), size and/or molecular mechanisms controlling HC sensitivity. This is one of the first studies investigating the ontogenetic development of the saccule and utricle in a vocal fish and how it potentially relates to auditory enhancement for acoustic communication.

Elastic force restricts growth of the murine utricle.

Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ's morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ's periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle's growth; interfering with Yap's activity reverses this effect.

ADAM10 and γ-secretase regulate sensory regeneration in the avian vestibular organs.

The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.

Co-regulation of the Notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles.

This work sought to determine the crosstalk between the Notch and Wnt signaling pathways in regulating supporting cell (SC) proliferation and hair cell (HC) regeneration in mouse utricles. We cultured postnatal day (P)3 and P60 mouse utricles, damaged the HCs with gentamicin, and treated the utricles with the γ-secretase inhibitor DAPT to inhibit the Notch pathway and with the Wnt agonist QS11 to active the Wnt pathway. We also used Sox2-CreER, Notch1-flox (exon 1), and Catnb-flox (exon 3) transgenic mice to knock out the Notch pathway and activate the Wnt pathway in Sox2+ SCs. Notch inhibition alone increased SC proliferation and HC number in both undamaged and damaged utricles. Wnt activation alone promoted SC proliferation, but the HC number was not significantly increased. Here we demonstrated the cumulative effects of Notch inhibition and Wnt activation in regulating SC proliferation and HC regeneration. Simultaneously inhibiting Notch and overexpressing Wnt led to significantly greater SC proliferation and greater numbers of HCs than manipulating either pathway alone. Similar results were observed in the transgenic mice. This study suggests that the combination of Notch inhibition and Wnt activation can significantly promote SC proliferation and increase the number of regenerated HCs in mouse utricle.

Temporal coupling between specifications of neuronal and macular fates of the inner ear.

The inner ear is a complex organ comprised of various specialized sensory organs for detecting sound and head movements. The timing of specification for these sensory organs, however, is not clear. Previous fate mapping results of the inner ear indicate that vestibular and auditory ganglia and two of the vestibular sensory organs, the utricular macula (UM) and saccular macula (SM), are lineage related. Based on the medial-lateral relationship where respective auditory and vestibular neuroblasts exit from the otic epithelium and the subsequent formation of the medial SM and lateral UM in these regions, we hypothesized that specification of the two lateral structures, the vestibular ganglion and the UM are coupled and likewise for the two medial structures, the auditory ganglion and the SM. We tested this hypothesis by surgically inverting the primary axes of the otic cup in ovo and investigating the fate of the vestibular neurogenic region, which had been spotted with a lipophilic dye. Our results showed that the laterally-positioned, dye-associated, vestibular ganglion and UM were largely normal in transplanted ears, whereas both auditory ganglion and SM showed abnormalities suggesting the lateral but not the medial-derived structures were mostly specified at the time of transplantation. Both of these results are consistent with a temporal coupling between neuronal and macular fate specifications.

Hearing Assessment in Zebrafish During the First Week Postfertilization.

The zebrafish (Danio rerio) is a valuable vertebrate model for human hearing disorders because of many advantages in genetics, embryology, and in vivo visualization. In this study, we investigated auditory function of zebrafish during the first week postfertilization using microphonic potential recording. Extracellular microphonic potentials were recorded from hair cells in the inner ear of wild-type AB and transgenic Et(krt4:GFP)(sqet4) zebrafish at 3, 5, and 7 days postfertilization in response to 20, 50, 100, 200, 300, and 400-Hz acoustic stimulation. We found that microphonic threshold significantly decreased with age in zebrafish. However, there was no significant difference of microphonic responses between wild-type and transgenic zebrafish, indicating that the transgenic zebrafish have normal hearing like wild-type zebrafish. In addition, we observed that microphonic threshold did not change with the recording electrode location. Furthermore, microphonic threshold increased significantly at all tested stimulus frequencies after displacement of the saccular otolith but only increased at low frequencies after displacement of the utricular otolith, showing that the saccule rather than the utricle plays the major role in larval zebrafish hearing. These results enhance our knowledge of early development of auditory function in zebrafish and the factors affecting hearing assessment with microphonic potential recording.

Spatial and Age-Dependent Hair Cell Generation in the Postnatal Mammalian Utricle.

Loss of vestibular hair cells is a common cause of balance disorders. Current treatment options for bilateral vestibular dysfunction are limited. During development, atonal homolog 1 (Atoh1) is sufficient and necessary for the formation of hair cells and provides a promising gene target to induce hair cell generation in the mammals. In this study, we used a transgenic mouse line to test the age and cell type specificity of hair cell induction in the postnatal utricle in mice. We found that forced Atoh1 expression in vivo can induce hair cell formation in the utricle from postnatal days 1 to 21, while the efficacy of hair cell induction is progressively reduced as the animals become older. In the utricle, the induction of hair cells occurs both within the sensory region and in cells in the transitional epithelium next to the sensory region. Within the sensory epithelium, the central region, known as the striola, is most subjective to the induction of hair cell formation. Furthermore, forced Atoh1 expression can promote proliferation in an age-dependent manner that mirrors the progressively reduced efficacy of hair cell induction in the postnatal utricle. These results suggest that targeting both cell proliferation and Atoh1 in the utricle striolar region may be explored to induce hair cell regeneration in mammals. The study also demonstrates the usefulness of the animal model that provides an in vivo Atoh1 induction model for vestibular regeneration studies.

Ontogenetic development of the auditory sensory organ in zebrafish (Danio rerio): changes in hearing sensitivity and related morphology.

Zebrafish (Danio rerio) is an important model organism in hearing research. However, data on the hearing sensitivity of zebrafish vary across different reports. In the present study, the hearing sensitivity of zebrafish was examined by analysing the auditory evoked potentials (AEPs) over a range of total lengths (TLs) from 12 to 46 mm. Morphological changes in the hair cells (HCs) of the saccule (the main auditory end organ) and their synapses with primary auditory neurons were investigated. The AEPs were detected up to a much higher frequency limit (12 kHz) than previously reported. No significant difference in the frequency response range was observed across the TL range examined. However, the AEP thresholds demonstrated both developmental improvement and age-related loss of hearing sensitivity. The changes in hearing sensitivity were roughly consistent with the morphological changes in the saccule including (1) the number and density of HCs, (2) the organization of stereocilia, and (3) the quantity of a main ribbon protein, Ribeye b. The results of this study established a clear baseline for the hearing ability of zebrafish and revealed that the changes in the saccule contribute to the observed changes in TL (age)-related hearing sensitivity.

Gene Expression by Mouse Inner Ear Hair Cells during Development.

Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes.