Parallel control of mechanosensory hair cell orientation by the PCP and Wnt pathways

ABSTRACTCell polarity plays a crucial role during development of vertebrates and invertebrates. Planar Cell Polarity (PCP) is defined as the coordinated polarity of cells within a tissue axis and is essential for processes such as gastrulation, neural tube closure or hearing. Wnt ligands can be instructive or permissive during PCP-dependent processes, and Wnt pathway mutants are often classified as PCP mutants due to the complexity and the similarities between their phenotypes. Our studies of the zebrafish sensory lateral line reveal that disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in PCP genes cause random orientations of hair cells, mutations in Wnt pathway members induce hair cells to adopt a concentric pattern. We show that PCP signaling is normal in hair cells of Wnt pathway mutants and that the concentric hair cell phenotype is due to altered organization of the surrounding support cells. Thus, the PCP and Wnt pathways work in parallel, as separate pathways to establish proper hair cell orientation. Our data suggest that coordinated support cell organization is established during the formation of lateral line primordia, much earlier than the appearance of hair cells. Together, these finding reveal that hair cell orientation defects are not solely explained by defects in PCP signaling and that some hair cell phenotypes warrant reevaluation.

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Par3 is essential for the establishment of planar cell polarity of inner ear hair cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816333116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4999-5008 ◽

Cited By ~ 11

Author(s):

Andre Landin Malt ◽

Zachary Dailey ◽

Julia Holbrook-Rasmussen ◽

Yuqiong Zheng ◽

Arielle Hogan ◽

...

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Cell Polarity ◽

Hair Cells ◽

Planar Cell Polarity ◽

Tissue Level ◽

Hair Bundle ◽

Nucleotide Exchange ◽

Genetic Mosaic ◽

Pcp Signaling

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3–GEF–Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.

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Using the Zebrafish Lateral Line to Understand the Roles of Mitochondria in Sensorineural Hearing Loss

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.628712 ◽

2021 ◽

Vol 8 ◽

Cited By ~ 1

Author(s):

Melanie Holmgren ◽

Lavinia Sheets

Keyword(s):

Cell Death ◽

Hearing Loss ◽

Hair Cell ◽

Sensorineural Hearing Loss ◽

Lateral Line ◽

Hair Cells ◽

High Energy ◽

Sensorineural Hearing ◽

Mitochondrial Activity ◽

Ototoxic Drugs

Hair cells are the mechanosensory receptors of the inner ear and can be damaged by noise, aging, and ototoxic drugs. This damage often results in permanent sensorineural hearing loss. Hair cells have high energy demands and rely on mitochondria to produce ATP as well as contribute to intracellular calcium homeostasis. In addition to generating ATP, mitochondria produce reactive oxygen species, which can lead to oxidative stress, and regulate cell death pathways. Zebrafish lateral-line hair cells are structurally and functionally analogous to cochlear hair cells but are optically and pharmacologically accessible within an intact specimen, making the zebrafish a good model in which to study hair-cell mitochondrial activity. Moreover, the ease of genetic manipulation of zebrafish embryos allows for the study of mutations implicated in human deafness, as well as the generation of transgenic models to visualize mitochondrial calcium transients and mitochondrial activity in live organisms. Studies of the zebrafish lateral line have shown that variations in mitochondrial activity can predict hair-cell susceptibility to damage by aminoglycosides or noise exposure. In addition, antioxidants have been shown to protect against noise trauma and ototoxic drug–induced hair-cell death. In this review, we discuss the tools and findings of recent investigations into zebrafish hair-cell mitochondria and their involvement in cellular processes, both under homeostatic conditions and in response to noise or ototoxic drugs. The zebrafish lateral line is a valuable model in which to study the roles of mitochondria in hair-cell pathologies and to develop therapeutic strategies to prevent sensorineural hearing loss in humans.

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Connectomics of the zebrafish's lateral-line neuromast reveals wiring and miswiring in a simple microcircuit

eLife ◽

10.7554/elife.33988 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 11

Author(s):

Eliot Dow ◽

Adrian Jacobo ◽

Sajjad Hossain ◽

Kimberly Siletti ◽

A J Hudspeth

Keyword(s):

Hair Cell ◽

Lateral Line ◽

Hair Cells ◽

Planar Cell Polarity ◽

Developmental Stages ◽

Notch Signaling Pathway ◽

Nerve Fibers ◽

Directional Sensitivity ◽

Efferent Nerve ◽

Polarity Gene

The lateral-line neuromast of the zebrafish displays a restricted, consistent pattern of innervation that facilitates the comparison of microcircuits across individuals, developmental stages, and genotypes. We used serial blockface scanning electron microscopy to determine from multiple specimens the neuromast connectome, a comprehensive set of connections between hair cells and afferent and efferent nerve fibers. This analysis delineated a complex but consistent wiring pattern with three striking characteristics: each nerve terminal is highly specific in receiving innervation from hair cells of a single directional sensitivity; the innervation is redundant; and the terminals manifest a hierarchy of dominance. Mutation of the canonical planar-cell-polarity gene vangl2, which decouples the asymmetric phenotypes of sibling hair-cell pairs, results in randomly positioned, randomly oriented sibling cells that nonetheless retain specific wiring. Because larvae that overexpress Notch exhibit uniformly oriented, uniformly innervating hair-cell siblings, wiring specificity is mediated by the Notch signaling pathway.

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Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling

The Journal of Cell Biology ◽

10.1083/jcb.201912071 ◽

2020 ◽

Vol 219 (10) ◽

Cited By ~ 1

Author(s):

Andre Landin Malt ◽

Arielle K. Hogan ◽

Connor D. Smith ◽

Maxwell S. Madani ◽

Xiaowei Lu

Keyword(s):

Hair Cell ◽

G Protein ◽

Cell Polarity ◽

Planar Cell Polarity ◽

Pi3k Pathway ◽

Sensory Epithelium ◽

Nucleotide Exchange ◽

Cell Orientation ◽

Complex Signals ◽

Heterotrimeric G Protein

In the mammalian cochlea, the planar cell polarity (PCP) pathway aligns hair cell orientation along the plane of the sensory epithelium. Concurrently, multiple cell intrinsic planar polarity (referred to as iPCP) modules mediate planar polarization of the hair cell apical cytoskeleton, including the kinocilium and the V-shaped hair bundle essential for mechanotransduction. How PCP and iPCP are coordinated during development and the roles of Wnt ligands in this process remain unresolved. Here we show that genetic blockade of Wnt secretion in the cochlear epithelium resulted in a shortened cochlear duct and misoriented and misshapen hair bundles. Mechanistically, Wnts stimulate Gi activity by regulating the localization of Daple, a guanine nucleotide exchange factor (GEF) for Gαi. In turn, the Gβγ complex signals through phosphoinositide 3-kinase (PI3K) to regulate kinocilium positioning and asymmetric localizations of a subset of core PCP proteins, thereby coordinating PCP and iPCP. Thus, our results identify a putative Wnt/heterotrimeric G protein/PI3K pathway for PCP regulation.

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Macrophages Respond Rapidly to Ototoxic Injury of Lateral Line Hair Cells but Are Not Required for Hair Cell Regeneration

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.613246 ◽

2021 ◽

Vol 14 ◽

Author(s):

Mark E. Warchol ◽

Angela Schrader ◽

Lavinia Sheets

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Lateral Line ◽

Hair Cells ◽

Control Fish ◽

Cell Regeneration ◽

Cellular Interactions ◽

Hair Cell Regeneration ◽

Genetic Ablation ◽

Larval Zebrafish

The sensory organs of the inner ear contain resident populations of macrophages, which are recruited to sites of cellular injury. Such macrophages are known to phagocytose the debris of dying cells but the full role of macrophages in otic pathology is not understood. Lateral line neuromasts of zebrafish contain hair cells that are nearly identical to those in the inner ear, and the optical clarity of larval zebrafish permits direct imaging of cellular interactions. In this study, we used larval zebrafish to characterize the response of macrophages to ototoxic injury of lateral line hair cells. Macrophages migrated into neuromasts within 20 min of exposure to the ototoxic antibiotic neomycin. The number of macrophages in the near vicinity of injured neuromasts was similar to that observed near uninjured neuromasts, suggesting that this early inflammatory response was mediated by “local” macrophages. Upon entering injured neuromasts, macrophages actively phagocytosed hair cell debris. The injury-evoked migration of macrophages was significantly reduced by inhibition of Src-family kinases. Using chemical-genetic ablation of macrophages before the ototoxic injury, we also examined whether macrophages were essential for the initiation of hair cell regeneration. Results revealed only minor differences in hair cell recovery in macrophage-depleted vs. control fish, suggesting that macrophages are not essential for the regeneration of lateral line hair cells.

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Directional sensitivity of hair cell afferents in the Octopus statocyst.

Journal of Experimental Biology ◽

10.1242/jeb.187.1.245 ◽

1994 ◽

Vol 187 (1) ◽

pp. 245-259 ◽

Cited By ~ 2

Author(s):

B U Budelmann ◽

R Williamson

Keyword(s):

Hair Cell ◽

Lateral Line ◽

Hair Cells ◽

Threshold Sensitivity ◽

Directional Sensitivity

Changes in threshold sensitivity of hair cell afferents of the macula and crista of the Octopus statocyst were analyzed when the hair cells were stimulated with sinusoidal water movements from different directions. The experiments indicate that cephalopod statocyst hair cells are directionally sensitive in a way that is similar to the responses of the hair cells of the vertebrate vestibular and lateral line systems, with the amplitude of the response changing according to the cosine of the angle by which the direction of the stimulus (the deflection of the ciliary bundle) deviates from the direction of the hair cell's morphological polarization.

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Vestibular and Auditory Hair Cell Regeneration Following Targeted Ablation of Hair Cells With Diphtheria Toxin in Zebrafish

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.721950 ◽

2021 ◽

Vol 15 ◽

Author(s):

Erin Jimenez ◽

Claire C. Slevin ◽

Luis Colón-Cruz ◽

Shawn M. Burgess

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Lateral Line ◽

Hair Cells ◽

Diphtheria Toxin ◽

Adult Zebrafish ◽

Hair Cell Regeneration ◽

Cell Ablation ◽

Aquatic Vertebrates

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

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Suppression of Inflammation Delays Hair Cell Regeneration and Functional Recovery Following Lateral Line Damage in Zebrafish Larvae

10.1101/2020.02.26.962753 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ru Zhang ◽

Xiao-Peng Liu ◽

Ya-Juan Li ◽

Ming Wang ◽

Lin Chen ◽

...

Keyword(s):

Hair Cell ◽

Functional Recovery ◽

Lateral Line ◽

Hair Cells ◽

Calcium Imaging ◽

Inflammatory Responses ◽

Early Stage ◽

Cell Regeneration ◽

Hair Cell Regeneration ◽

Cochlear Hair Cells

AbstractBackgroundHuman cochlear hair cells cannot spontaneously regenerate after loss. In contrast, those in fish and amphibians have a remarkable ability to regenerate after damaged. Previous studies focus on signaling mechanisms of hair cell regeneration, such as Wnt and Notch signals but seldom on the fact that the beginning of regeneration is accompanied by a large number of inflammatory responses. The detailed role of this inflammation in hair cell regeneration is still unknown. In addition, there is no appropriate behavioral method to quantitatively evaluate the functional recovery of lateral line hair cells after regeneration.ResultsIn this study, we found that when inflammation was suppressed, the regeneration of lateral line hair cells and the recovery of the rheotaxis of the larvae were significantly delayed. Calcium imaging showed that the function of the neuromasts in the inflammation-inhibited group was weaker than that in the non-inflammation-inhibited group at the Early Stage of regeneration, and returned to normal at the Late Stage. Calcium imaging also revealed the cause of the mismatch between the function and quantity during regeneration.ConclusionsOur results, meanwhile, suggest that suppressing inflammation delays hair cell regeneration and functional recovery when hair cells are damaged. This study may provide a new knowledge for how to promote hair cell regeneration and functional recovery in adult mammals.

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Neural transduction in Xenopus laevis lateral line system

Journal of Neurophysiology ◽

10.1152/jn.1978.41.2.432 ◽

1978 ◽

Vol 41 (2) ◽

pp. 432-444 ◽

Cited By ~ 39

Author(s):

D. Strelioff ◽

V. Honrubia

Keyword(s):

Hair Cell ◽

Lateral Line ◽

Hair Cells ◽

Water Movement ◽

Neural Response ◽

Neural Responses ◽

Cell Systems ◽

Afferent Fibers ◽

Neural Excitation ◽

Electrical Stimuli

1. The process of neural excitation in hair cell systems was studied in an in vitro preparation of the Xenopus laevis (African clawed toad) lateral line organ. A specially designed stimulus chamber was used to apply accurately controlled pressure, water movement, or electrical stimuli, and to record the neural responses of the two afferent fibers innervating each organ or stitch. The objective of the study was to determine the characteristics of the neural responses to these stimuli, and thus gain insight into the transduction process. 2. A sustained deflection of the hair cell cilia due to a constant flow of water past the capula resulted in a maintained change in the mean firing rate (MFR) of the afferent fibers. The data also demonstrated that the neural response was proportional to the velocity of the water flow and indicated that both deflection and movement of the cilia were the effective physiological stimuli for this hair cell system. 3. The preparations responded to sinusoidal water movements (past the capula) over the entire frequency range of the stimulus chamber, 0.1-130 Hz, and were most sensitive between 10 and 40 Hz. The variation of the MFR and the percent modulation indicated that the average dynamic range of each organ was 23.5 dB. 4. The thresholds, if any, for sustained pressure changes and for sinusoidal pressure variations in the absence of water movements were very high. Due to the limitations of the stimulus chamber it was not possible to generate pressure stimuli of sufficient magnitude to elicit a neural response without also generating suprathreshold water-movement stimuli. Sustained pressures had no detectable effect on the neural response to water-movement stimuli. 5. The preparations were very sensitive to electrical potentials applied across the toad skin on which the hair cells were located. Potentials which made the ciliated surfaces of the hair cells positive with respect to their bases increased the MFR of the fibers, whereas negative potentials decreased it. The responses to sinusoidal electrical stimuli were similar to responses to water-movement stimuli with respect to frequency and dynamic ranges. Thresholds as low as 100 muV peak to peak (p-p) for 16-Hz stimuli were found. 6. The characteristics of the neural responses to electrical stimulation as well as supporting data obtained from the studies of the effects of anoxia on the evoked responses indicate that the electrical stimulus acts on the hair cells or on the synapses, rather than directly on the nerve fibers. This finding suggests that receptor potentials or their associated currents play an important role in the process of neural excitation in hair cell systems.

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