Fish communicate with water flow to enhance a school's social network.

Schooling fish rely on a social network created through signaling between its members to interact with their environment. Previous studies have established that vision is necessary for schooling and that flow sensing by the lateral line system may aid in a school's cohesion. However, it remains unclear to what extent flow provides a channel of communication between schooling fish. Based on kinematic measurements of the speed and heading of schooling tetras (Petitella rhodostoma), we found that compromising the lateral line by chemical treatment reduced the mutual information between individuals by ∼13%. This relatively small reduction in pairwise communication propagated through schools of varying size to reduce the degree and connectivity of the social network by more than half. Treated schools additionally showed more than twice the spatial heterogeneity of fish with unaltered flow sensing. These effects were much more substantial than the changes that we measured in the nearest-neighbor distance, speed and intermittency of individual fish by compromising flow sensing. Therefore, flow serves as a valuable supplement to visual communication in a manner that is revealed through a school's network properties.

The Silverjaw Minnow, Ericymba buccata: An Extraordinary Lateral Line System and its Contribution to Prey Detection.

Fishes use their mechanosensory lateral line (LL) system to detect local water flows in different behavioral contexts, including the detection of prey. The LL system is comprised of neuromast receptor organs on the skin (superficial neuromasts) and within bony canals (canal neuromasts). Most fishes have one cranial LL canal phenotype, but the silverjaw minnow (Ericymba buccata) has two: narrow canals dorsal and caudal to the eye and widened canals ventral to the eye and along the mandible. The ventrally directed widened LL canals have been hypothesized to be an adaptation for detection of their benthic prey. Multiple morphological methods were used to describe the narrow and widened canals and canal neuromasts in detail. The primary distribution of hundreds of superficial neuromasts and taste buds ventral to the eye and on the mandible (described here for the first time) suggests additional sensory investment for detecting flow and chemical stimuli emanating from benthic prey. The hypothesis that the LL system mediates prey localization was tested by measuring five parameters in behavioral trials in which the combination of sensory modalities available to fish was manipulated (four experimental treatments). Fish detected and localized prey regardless of available sensory modalities and they were able to detect prey in the dark in the absence of LL input (LL ablation with neomycin sulfate) revealing that chemoreception was sufficient to mediate benthic prey detection, localization, and consumption. However, elimination of LL input resulted in a change in the angle of approach to live (mobile) prey even when visual input was available, suggesting that mechanosensory input contributes to the successful detection and localization of prey. The results of this study demonstrate that the extraordinary LL canal system of the silverjaw minnow, in addition to the large number of superficial neuromasts, and the presence of numerous extraoral taste buds, likely represent adaptations for multimodal integration of sensory inputs contributing to foraging behavior in this species. The morphological and behavioral results of this study both suggest that this species would be an excellent model for future comparative structural and functional studies of sensory systems in fishes.

Tadpoles rely on mechanosensory stimuli for communication when visual capabilities are poor.

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species.

Real-time position and pose prediction for a self-propelled undulatory swimmer in 3D space with artificial lateral line system.

This study aims to investigate the feasibility of using an artificial lateral line (ALL) system for predicting the real-time position and pose of an undulating swimmer with Carangiform swimming patterns. We established a 3D computational fluid dynamics simulation to replicate the swimming dynamics of a freely swimming mackerel under various motion parameters, calculating the corresponding pressure fields. Using the simulated lateral line data, we trained an artificial neural network to predict the centroid coordinates and orientation of the swimmer. A comprehensive analysis was further conducted to explore the impact of sensor quantity, distribution, noise amplitude and sampling intervals of the ALL array on predicting performance. Additionally, to quantitatively assess the reliability of the localization network, we trained another neural network to evaluate error magnitudes for different input signals. These findings provide valuable insights for guiding future research on mutual sensing and schooling in underwater robotic fish.

The role of vision and lateral line sensing for schooling in giant danios (Devario aequipinnatus).

Schooling is a collective behavior that relies on a fish's ability to sense and respond to the other fish around it. Previous work has identified 'rules' of schooling - attraction to neighbors that are far away, repulsion from neighbors that are too close and alignment with neighbors at the correct distance - but we do not understand well how these rules emerge from the sensory physiology and behavior of individual fish. In particular, fish use both vision and their lateral lines to sense each other, but it is unclear how much they rely on information from these sensory modalities to coordinate schooling behavior. To address this question, we studied how the schooling of giant danios (Devario aequipinnatus) changes when they are unable to see or use their lateral lines. We found that giant danios were able to school without their lateral lines but did not school in darkness. Surprisingly, giant danios in darkness had the same attraction properties as fish in light when they were in close proximity, indicating that they could sense nearby fish with their lateral lines. However, they were not attracted to more distant fish, suggesting that long-distance attraction through vision is important for maintaining a cohesive school. These results help us expand our understanding of the roles that vision and the lateral line play in the schooling of some fish species.

Anion efflux mediates transduction in the hair cells of the zebrafish lateral line.

Hair cells are the principal sensory receptors of the vertebrate auditory system, where they transduce sounds through mechanically gated ion channels that permit cations to flow from the surrounding endolymph into the cells. The lateral line of zebrafish has served as a key model system for understanding hair cell physiology and development, often with the belief that these hair cells employ a similar transduction mechanism. In this study, we demonstrate that these hair cells are exposed to an unregulated external environment with cation concentrations that are too low to support transduction. Our results indicate that hair cell excitation is instead mediated by a substantially different mechanism involving the outward flow of anions. Further investigation of hair cell transduction in a diversity of sensory systems and species will likely yield deep insights into the physiology of these unique cells.

Efferent axons in the zebrafish lateral line degenerate following sensory hair cell ablation.

The zebrafish lateral line is a frequently used model to study the mechanisms behind peripheral neuronal innervation of sensory organs and the regeneration thereof. The lateral line system consists of neuromasts, a cluster of protruding hair cells, which are innervated by sensory afferent and modulatory efferent neurons. These flow-sensing hair cells are similar to the hair cells in the mammalian ear. Though, while hair cell loss in humans is irreversible, the zebrafish neuromasts are regarded as the fastest regenerating structure in vertebrates, making them an ideal model to study regeneration. However, one component of the lateral line system, the efferent projections, has largely been omitted in regenerative studies. Here, for the first time, we bring insights into the fate of efferent axons during ablation and regeneration of the hair cells in the zebrafish lateral line. Our behavioral analysis showed functional recovery of hair cells and sensory transmission within 48 h and their regeneration were in line with previous studies. Analysis of the inhibitory efferent projections revealed that in approximately half the cases the inhibitory efferent axons degenerated, which was never observed for the sensory afferent axons. Quantification of hair cells following ablation suggests that the presence of mature hair cells in the neuromast may prevent axon degeneration. Within 120 h, degenerated efferent axons regenerated along the axonal tract of the lateral line. Reanalysis of published single cell neuromast data hinted to a role for Bdnf in the survival of efferent axons. However, sequestering Bdnf, blocking the Trk-receptors, and inhibiting the downstream ERK-signaling, did not induce axon degeneration, indicating that efferent survival is not mediated through neurotrophic factors. To further explore the relation between hair cells and efferent projections, we generated atoh1a mutants, where mature hair cells never form. In larvae lacking hair cells, inhibitory efferent projections were still present, following the tract of the sensory afferent without displaying any innervation. Our study reveal the fate of efferent innervation following hair cell ablation and provide insights into the inherent differences in regeneration between neurons in the peripheral and central nervous system.

In vivo investigation of mitochondria in lateral line afferent neurons and hair cells.

To process sensory stimuli, intense energy demands are placed on hair cells and primary afferents. Hair cells must both mechanotransduce and maintain pools of synaptic vesicles for neurotransmission. Furthermore, both hair cells and afferent neurons must continually maintain a polarized membrane to propagate sensory information. These processes are energy demanding and therefore both cell types are critically reliant on mitochondrial health and function for their activity and maintenance. Based on these demands, it is not surprising that deficits in mitochondrial health can negatively impact the auditory and vestibular systems. In this review, we reflect on how mitochondrial function and dysfunction are implicated in hair cell-mediated sensory system biology. Specifically, we focus on live imaging approaches that have been applied to study mitochondria using the zebrafish lateral-line system. We highlight the fluorescent dyes and genetically encoded biosensors that have been used to study mitochondria in lateral-line hair cells and afferent neurons. We then describe the impact this in vivo work has had on the field of mitochondrial biology as well as the relationship between mitochondria and sensory system development, function, and survival. Finally, we delineate the areas in need of further exploration. This includes in vivo analyses of mitochondrial dynamics and biogenesis, which will round out our understanding of mitochondrial biology in this sensitive sensory system.

Asymmetric mechanotransduction by hair cells of the zebrafish lateral line.

In the lateral line system, water motion is detected by neuromast organs, fundamental units that are arrayed on a fish's surface. Each neuromast contains hair cells, specialized mechanoreceptors that convert mechanical stimuli, in the form of water movement, into electrical signals. The orientation of hair cells' mechanosensitive structures ensures that the opening of mechanically gated channels is maximal when deflected in a single direction. In each neuromast organ, hair cells have two opposing orientations, enabling bi-directional detection of water movement. Interestingly, Tmc2b and Tmc2a proteins, which constitute the mechanotransduction channels in neuromasts, distribute asymmetrically so that Tmc2a is expressed in hair cells of only one orientation. Here, using both in vivo recording of extracellular potentials and calcium imaging of neuromasts, we demonstrate that hair cells of one orientation have larger mechanosensitive responses. The associated afferent neuron processes that innervate neuromast hair cells faithfully preserve this functional difference. Moreover, Emx2, a transcription factor required for the formation of hair cells with opposing orientations, is necessary to establish this functional asymmetry within neuromasts. Remarkably, loss of Tmc2a does not impact hair cell orientation but abolishes the functional asymmetry as measured by recording extracellular potentials and calcium imaging. Overall, our work indicates that oppositely oriented hair cells within a neuromast employ different proteins to alter mechanotransduction to sense the direction of water motion.

Structural and functional evolution of the mechanosensory lateral line system of fishesa).

The mechanosensory lateral line system is the flow sensing system present in all 34 000+ species of fishes. Its neuromast receptor organs, located on the skin or in bony canals on the head and tubed scales on the trunk, respond to the near field component of acoustic stimuli as well as short range, low frequency (0-200 Hz) water flows of biotic and abiotic origin. Here, I discuss the genesis of my research career and its focus on the structural and functional evolution of the lateral line system among a wide taxonomic range of fishes including those from different aquatic habitats (tropical lakes to coral reefs and the deep sea). I discuss the importance of investigating structure before function, using investigations in my laboratory that had unexpected outcomes, as well as the role of serendipity in the evolution of a career and in the nature of scientific discovery.

Prolonged Dexamethasone Exposure Enhances Zebrafish Lateral-Line Regeneration But Disrupts Mitochondrial Homeostasis and Hair Cell Function.

The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 h. Consistent with previous work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed hyperpolarized mitochondrial membrane potentials (ΔΨm) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.

Evolutionary convergence of a neural mechanism in the cavefish lateral line system.

Animals can evolve dramatic sensory functions in response to environmental constraints, but little is known about the neural mechanisms underlying these changes. The Mexican tetra, Astyanax mexicanus, is a leading model to study genetic, behavioral, and physiological evolution by comparing eyed surface populations and blind cave populations. We compared neurophysiological responses of posterior lateral line afferent neurons and motor neurons across A. mexicanus populations to reveal how shifts in sensory function may shape behavioral diversity. These studies indicate differences in intrinsic afferent signaling and gain control across populations. Elevated endogenous afferent activity identified a lower response threshold in the lateral line of blind cavefish relative to surface fish leading to increased evoked potentials during hair cell deflection in cavefish. We next measured the effect of inhibitory corollary discharges from hindbrain efferent neurons onto afferents during locomotion. We discovered that three independently derived cavefish populations have evolved persistent afferent activity during locomotion, suggesting for the first time that partial loss of function in the efferent system can be an evolutionary mechanism for neural adaptation of a vertebrate sensory system.

Functional and ultrastructural analysis of reafferent mechanosensation in larval zebrafish.

All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animal's proximity, which need to be distinguishable from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body. In this study, we characterize in detail how hair cells-the receptor cells of the lateral line-in zebrafish larvae discriminate between such reafferent and exafferent signals. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies (copies of the motor command that cancel out self-generated reafferent stimulation during locomotion) and that dopaminergic signals from the hypothalamus may have a role in threshold modulation, both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts. We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.

The early development and physiology of Xenopus laevis tadpole lateral line system.

Xenopus laevis has a lateral line mechanosensory system throughout its full life cycle, and a previous study on prefeeding stage tadpoles revealed that it may play a role in motor responses to both water suction and water jets. Here, we investigated the physiology of the anterior lateral line system in newly hatched tadpoles and the motor outputs induced by its activation in response to brief suction stimuli. High-speed videoing showed tadpoles tended to turn and swim away when strong suction was applied close to the head. The lateral line neuromasts were revealed by using DASPEI staining, and their inactivation with neomycin eliminated tadpole motor responses to suction. In immobilized preparations, suction or electrically stimulating the anterior lateral line nerve reliably initiated swimming but the motor nerve discharges implicating turning was observed only occasionally. The same stimulation applied during ongoing fictive swimming produced a halting response. The anterior lateral line nerve showed spontaneous afferent discharges at rest and increased activity during stimulation. Efferent activities were only recorded during tadpole fictive swimming and were largely synchronous with the ipsilateral motor nerve discharges. Finally, calcium imaging identified neurons with fluorescence increase time-locked with suction stimulation in the hindbrain and midbrain. A cluster of neurons at the entry point of the anterior lateral line nerve in the dorsolateral hindbrain had the shortest latency in their responses, supporting their potential sensory interneuron identity. Future studies need to reveal how the lateral line sensory information is processed by the central circuit to determine tadpole motor behavior.NEW & NOTEWORTHY We studied Xenopus tadpole motor responses to anterior lateral line stimulation using high-speed videos, electrophysiology and calcium imaging. Activating the lateral line reliably started swimming. At high stimulation intensities, turning was observed behaviorally but suitable motor nerve discharges were seen only occasionally in immobilized tadpoles. Suction applied during swimming produced a halting response. We analyzed afferent and efferent activities of the tadpole anterior lateral line nerve and located sensory interneurons using calcium imaging.

Corollary discharge enables proprioception from lateral line sensory feedback.

Animals modulate sensory processing in concert with motor actions. Parallel copies of motor signals, called corollary discharge (CD), prepare the nervous system to process the mixture of externally and self-generated (reafferent) feedback that arises during locomotion. Commonly, CD in the peripheral nervous system cancels reafference to protect sensors and the central nervous system from being fatigued and overwhelmed by self-generated feedback. However, cancellation also limits the feedback that contributes to an animal's awareness of its body position and motion within the environment, the sense of proprioception. We propose that, rather than cancellation, CD to the fish lateral line organ restructures reafference to maximize proprioceptive information content. Fishes' undulatory body motions induce reafferent feedback that can encode the body's instantaneous configuration with respect to fluid flows. We combined experimental and computational analyses of swimming biomechanics and hair cell physiology to develop a neuromechanical model of how fish can track peak body curvature, a key signature of axial undulatory locomotion. Without CD, this computation would be challenged by sensory adaptation, typified by decaying sensitivity and phase distortions with respect to an input stimulus. We find that CD interacts synergistically with sensor polarization to sharpen sensitivity along sensors' preferred axes. The sharpening of sensitivity regulates spiking to a narrow interval coinciding with peak reafferent stimulation, which prevents adaptation and homogenizes the otherwise variable sensor output. Our integrative model reveals a vital role of CD for ensuring precise proprioceptive feedback during undulatory locomotion, which we term external proprioception.

Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish.

Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.

Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behavior.

Social behavior is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behavior, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behavior. Overall, these results suggest that the antagonistic association between repetitive and social-like behaviors is deeply shared from teleosts through mammals.

Learning hydrodynamic signatures through proprioceptive sensing by bioinspired swimmers.

Objects moving in water or stationary objects in streams create a vortex wake. Such vortex wakes encode information about the objects and the flow conditions. Underwater robots that often function with constrained sensing capabilities can benefit from extracting this information from vortex wakes. Many species of fish do exactly this, by sensing flow features using their lateral lines as part of their multimodal sensing. To replicate such capabilities in robots, significant research has been devoted to developing artificial lateral line sensors that can be placed on the surface of a robot to detect pressure and velocity gradients. We advance an alternative view of embodied sensing in this paper; the kinematics of a swimmer's body in response to the hydrodynamic forcing by the vortex wake can encode information about the vortex wake. Here we show that using artificial neural networks that take the angular velocity of the body as input, fish-like swimmers can be trained to label vortex wakes which are hydrodynamic signatures of other moving bodies and thus acquire a capability to 'blindly' identify them.

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.

Emx2 regulates hair cell rearrangement but not positional identity within neuromasts.

Each hair cell (HC) precursor of zebrafish neuromasts divides to form two daughter HCs of opposite hair bundle orientations. Previously, we showed that transcription factor Emx2, expressed in only one of the daughter HCs, generates this bidirectional HC pattern (Jiang et al., 2017). Here, we asked whether Emx2 mediates this effect by changing location of hair bundle establishment or positions of HCs since daughter HCs are known to switch positions with each other. We showed this HC rearrangement, redefined as two processes named Rock and Roll, is required for positional acquisition of HCs. Apical protrusion formation of nascent HCs and planar polarity signaling are both important for the Rock and Roll. Emx2 facilitates Rock and Roll by delaying apical protrusion of its nascent HCs but it does not determine HCs' ultimate positions, indicating that Emx2 mediates bidirectional HC pattern by changing the location where hair bundle is established in HCs.