Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity.

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable-and easily accessible-tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

Age-related changes in the zebrafish and killifish inner ear and lateral line.

Age-related hearing loss (ARHL) is a debilitating disorder for millions worldwide. While there are multiple underlying causes of ARHL, one common factor is loss of sensory hair cells. In mammals, new hair cells are not produced postnatally and do not regenerate after damage, leading to permanent hearing impairment. By contrast, fish produce hair cells throughout life and robustly regenerate these cells after toxic insult. Despite these regenerative abilities, zebrafish show features of ARHL. Here, we show that aged zebrafish of both sexes exhibited significant hair cell loss and decreased cell proliferation in all inner ear epithelia (saccule, lagena, utricle). Ears from aged zebrafish had increased expression of pro-inflammatory genes and significantly more macrophages than ears from young adult animals. Aged zebrafish also had fewer lateral line hair cells and less cell proliferation than young animals, although lateral line hair cells still robustly regenerated following damage. Unlike zebrafish, African turquoise killifish (an emerging aging model) only showed hair cell loss in the saccule of aged males, but both sexes exhibit age-related changes in the lateral line. Our work demonstrates that zebrafish exhibit key features of auditory aging, including hair cell loss and increased inflammation. Further, our finding that aged zebrafish have fewer lateral line hair cells yet retain regenerative capacity, suggests a decoupling of homeostatic hair cell addition from regeneration following acute trauma. Finally, zebrafish and killifish show species-specific strategies for lateral line homeostasis that may inform further comparative research on aging in mechanosensory systems.

Lateral line system diversification during the early stages of ecological speciation in cichlid fish.

BACKGROUND: The mechanosensory lateral line system is an important sensory modality in fishes, informing multiple behaviours related to survival including finding food and navigating in dark environments. Given its ecological importance, we may expect lateral line morphology to be under disruptive selection early in the ecological speciation process. Here we quantify the lateral line system morphology of two ecomorphs of the cichlid fish Astatotilapia calliptera in crater Lake Masoko that have diverged from common ancestry within the past 1,000 years. RESULTS: Based on geometric morphometric analyses of CT scans, we show that the zooplanktivorous benthic ecomorph that dominates the deeper waters of the lake has large cranial lateral line canal pores, relative to those of the nearshore invertebrate-feeding littoral ecomorph found in the shallower waters. In contrast, fluorescence imaging revealed no evidence for divergence between ecomorphs in the number of either superficial or canal neuromasts. We illustrate the magnitude of the variation we observe in Lake Masoko A. calliptera in the context of the neighbouring Lake Malawi mega-radiation that comprises over 700 species. CONCLUSIONS: These results provide the first evidence of divergence in this often-overlooked sensory modality in the early stages of ecological speciation, suggesting that it may have a role in the broader adaptive radiation process.

Macrophages enhance regeneration of lateral line neuromast derived from interneuromast cells through TGF-ß in zebrafish.

Macrophages play a pivotal role in the response to injury, contributing significantly to the repair and regrowth of damaged tissues. The external lateral line system in aquatic organisms offers a practical model for studying regeneration, featuring interneuromast cells connecting sensory neuromasts. Under normal conditions, these cells remain dormant, but their transformation into neuromasts occurs when overcoming inhibitory signals from Schwann cells and posterior lateral line nerves. The mechanism enabling interneuromast cells to evade inhibition by Schwann cells remains unclear. Previous observations suggest that macrophages physically interact with neuromasts, nerves, and Schwann cells during regeneration. This interaction leads to the regeneration of neuromasts in a subset of zebrafish with ablated neuromasts. To explore whether macrophages achieve this effect through secreted cytokines, we conducted experiments involving tail amputation in zebrafish larvae and tested the impact of cytokine inhibitors on neuromast regeneration. Most injured larvae remarkably regenerated a neuromast within 4 days post-amputation. Intriguingly, removal of macrophages and inhibition of the anti-inflammatory cytokine transforming growth factor-beta (TGF-ß) significantly delayed neuromast regeneration. Conversely, inhibition of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) had minor effects on the regeneration process. This study provides insights into how macrophages activate interneuromast cells, elucidating the pathways underlying neuromast regeneration.

Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.

RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

HIF signaling overactivation inhibits lateral line neuromast development through Wnt in zebrafish.

The lateral line is critical for prey detection, predator avoidance, schooling, and rheotaxis behavior in fish. As similar to hair cells in the mammalian inner ear, the lateral line sensory organ called neuromasts is a popular model for hair cell regeneration. However, the mechanism of lateral line development has not been fully understood. In this study, we showed for the first time that hypoxia-inducible factor (HIF) signaling is involved in lateral line development in zebrafish. hif1ab and epas1b were highly expressed in neuromasts during lateral line development. Hypoxia response induced by a prolyl hydroxylase domain-containing proteins (PHD) inhibitor treatment or vhl gene knockout significantly reduced hair cells and support cells in neuromast during lateral line development. In addition, inhibition of Hif-1α or Epas1 could partially rescue hair cells in the larvae with increased HIF activity, respectively. Moreover, the support cell proliferation and the expression of Wnt target genes decreased in vhl mutants which suggests that Wnt signaling mediated the role of HIF signaling in lateral line development. Collectively, our results demonstrate that HIF signaling overactivation inhibits lateral line development in zebrafish and suggest that inhibition of HIF signaling might be a potential therapeutic method for hair cell death.

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.

Radial polarity in the first cranial neuromast of selected teleost fishes.

The neuromast is a sensory structure of the lateral line system in aquatic vertebrates, which consists of hair cells and supporting cells. Hair cells are mechanosensory cells, generally arranged with bidirectional polarity. Here, we describe a neuromast with hair cells arranged radially instead of bidirectionally in the first cranial neuromast of four teleost species: red seabream (Pagrus major), spotted halibut (Verasper variegatus), brown sole (Pseudopleuronectes herzensteini), and marbled sole (Pseudopleuronectes yokohamae). In these four species, this polarity was identified only in the first cranial neuromast, where it appeared at the rostral edge of the otic vesicle before hatching. We investigated the initial appearance and fate of this unique neuromast using scanning electron microscopy. We also assessed characteristics of radial neuromast pertaining to morphogenesis, development, and innervation using a vital fluorescent marker and immunohistochemistry in V. variegatus. The kinocilium initially appears at the center of each hair cell, then moves to its outer perimeter to form radial polarity by around 7 days postfertilization. However, hair cells arranged radially disappear about 15 days after hatching. This is followed by the appearance of bidirectionally arranged hair cells, indicating that polarity replacement from radial to bidirectional has occurred. In P. herzensteini, both afferent and efferent synapses between the nerve fibers and hair cells were observed by transmission electron microscopy, suggesting that radial neuromast is functional. Our discovery suggests that neuromasts with radial polarity could enable larval fish to assimilate multiaxial stimuli during this life stage, potentially assisting them in detecting small water vibrations or water pressure changes.

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.

Sublethal effects of methylmercury on lateral line sensory and ion-regulatory functions in zebrafish embryos.

Methylmercury can interfere with the normal functioning of the nervous system, causing a variety of behavioral and physiological changes in fish. However, the influence of MeHg on the lateral line sensory and ion-regulatory functions of fish is not clear. Zebrafish embryos were utilized as a model to address this question. After exposure to water-borne MeHg (5, 10, 50, or 100 ppb) for 96 h (4-100 h post-fertilization), the survival rate declined by ca. 50 % at 100 ppb. However, MeHg at sublethal concentrations delayed hatching and decreased heart rates and body length. As to effects on the lateral line sensory system, MeHg at ≥10 ppb decreased the number of hair cells and impaired hair bundles and Ca2+-mediated mechanical transduction. As to ion regulation, MeHg at ≥10 ppb decreased the densities of skin stem cells and ionocytes, leading to declines in ion (Na+, K+, and Ca2+) contents and H+/NH4+ excretion levels. A gene expression analysis also revealed declines in messenger RNA levels of several ion-regulatory genes (ncc2b, trpv6v1a, trpv5/6, ncx1b, and rhcg1). This study demonstrated that the lateral line sensory and ion regulatory functions of fish are extremely sensitive to MeHg.

Toxic effects of polystyrene nanoparticles on the development, escape locomotion, and lateral-line sensory function of zebrafish embryos.

Environmental pollution by micro- and nanosized plastic particles is a potential threat to aquatic animals. Polystyrene is one of the most common plastic particles in aquatic environments. Previous studies found that polystyrene nanoparticles (PNs) can penetrate the integument and accumulate in the organs of fish embryos. However, the potential impacts of PNs on fish embryos are not fully understood. To investigate this issue, zebrafish embryos were exposed to different concentrations (10, 25, and 50 mg/L) of PNs (25 nm) for 96 h (4-100 h post-fertilization), and various endpoints were examined, including developmental morphology (body length, sizes of the eyes, otic vesicles, otoliths, pericardial cavity, and yolk sac), locomotion (touch-evoked escape response and spinal motor neurons), and lateral-line function (hair cell number and hair bundle number). Exposure to 50 mg/L of PNs resulted in significant adverse effects across all endpoints studied, indicating that embryonic development was severely disrupted, and both locomotion and sensory function were impaired. However, at 25 mg/L of PNs, only locomotion and sensory function were significantly affected. The effects were insignificant in all examined endpoints at 10 mg/L of PNs. Transcript levels of several marker genes for neuronal function and eye development were suppressed after treatment. Exposure to fluorescent PNs showed that they accumulated in various organs including, the eyes, gills, blood vessels, gallbladder, gut, and lateral line neuromasts. Overall, this study suggests that short-term exposure to a high concentration of PNs can threaten fish survival by impairing embryonic development, locomotion performance, and mechanical sensory function.

A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line.

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as 'surfacing'. Little is known about the sensory basis for this underappreciated behavior of larval fish. A strong candidate is the mechanosensory lateral line, a hair cell-based sensory system that detects hydrodynamic information from sources such as water currents, predators, prey and surface waves. However, a role for the lateral line in mediating initial inflation of the swim bladder has not been reported. To explore the connection between the lateral line and surfacing, we used a genetic mutant (lhfpl5b-/-) that renders the zebrafish lateral line insensitive to mechanical stimuli. We observed that approximately half of these lateral line mutants over-inflate their swim bladders during initial inflation and become positively buoyant. Thus, we hypothesized that larval zebrafish use their lateral line to moderate interactions with the air-water interface during surfacing to regulate swim bladder inflation. To test the hypothesis that lateral line defects are responsible for swim bladder over-inflation, we showed that exogenous air is required for the hyperinflation phenotype and transgenic rescue of hair cell function restores normal inflation. We also found that chemical ablation of anterior lateral line hair cells in wild-type larvae causes hyperinflation. Furthermore, we show that manipulation of lateral line sensory information results in abnormal inflation. Finally, we report spatial and temporal differences in the surfacing behavior between wild-type and lateral line mutant larvae. In summary, we propose a novel sensory basis for achieving neutral buoyancy where larval zebrafish use their lateral line to sense the air-water interface and regulate initial swim bladder inflation.

A study on flow field characteristics of a self-propelled robot fish approaching static obstacles based on artificial lateral line.

To perceive the static obstacles in still water, the flow field characteristics of a self-propelled robot fish approaching static obstacles were studied based on artificial lateral line (ALL). The pressure distribution on the fish body surface was calculated with different separation between the robot fish and the obstacle boundary, obstacle size and undulating frequency. Subsequently, an ALL system was established and five obstacle perception models were studied to analyze the perceptual characteristics of the ALL. Finally, the experiments were conducted to further reveal the effects of obstacles and motion parameters on the body surface pressure of robot fish. The results indicate that the obstacles have a significant effect on the pressure distribution of the surface of the fish body. Namely the parameters of separation, obstacle size and undulating frequency will affect the peak value of the amplitude envelope of the pressure signals. The obstacle size and distance between the obstacles can be predicted using the time parameters of the amplitude envelope of the pressure signals. Moreover, the self-propelled robot fish with a medium undulating frequency approach to the large obstacles with small separation has better perceptual performance. The findings could offer some insight into understanding the perception of complex underwater environment based on ALL.

Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells.

Hair cells of the inner ear are particularly sensitive to changes in mitochondria, the subcellular organelles necessary for energy production in all eukaryotic cells. There are over 30 mitochondrial deafness genes, and mitochondria are implicated in hair cell death following noise exposure, aminoglycoside antibiotic exposure, as well as in age-related hearing loss. However, little is known about the basic aspects of hair cell mitochondrial biology. Using hair cells from the zebrafish lateral line as a model and serial block-face scanning electron microscopy, we have quantifiably characterized a unique hair cell mitochondrial phenotype that includes (1) a high mitochondrial volume and (2) specific mitochondrial architecture: multiple small mitochondria apically, and a reticular mitochondrial network basally. This phenotype develops gradually over the lifetime of the hair cell. Disrupting this mitochondrial phenotype with a mutation in opa1 impacts mitochondrial health and function. While hair cell activity is not required for the high mitochondrial volume, it shapes the mitochondrial architecture, with mechanotransduction necessary for all patterning, and synaptic transmission necessary for the development of mitochondrial networks. These results demonstrate the high degree to which hair cells regulate their mitochondria for optimal physiology and provide new insights into mitochondrial deafness.

Our ability to perceive sounds relies on tiny cells deep inside our ears which can convert vibrations into the electrical signals that our brain is able to decode. These 'hair cells' sport a small tuft of short fibers on one of their ends that can move in response to pressure waves. The large amount of energy required for this activity is provided by the cells' mitochondria, the small internal compartments that act as cellular powerhouses. In fact, reducing mitochondrial function in hair cells can lead to hearing disorders. Mitochondria are often depicted as being bean-like, but they can actually adopt different shapes based on the level of energy they need to produce. Despite this link between morphology and function, little is known about what mitochondria look like in hair cells. Filling this knowledge gap is necessary to understand how these structures support hair cells and healthy hearing. To address this question, McQuate et al. turned to zebrafish, as these animals detect vibrations in water through easily accessible hair cells on their skin that work just like the ones in the mammalian ear. Obtaining and analysing series of 3D images from a high-resolution microscope revealed that hair cells are more densely populated with mitochondria than other cell types. Mitochondrial organisation was also strikingly different. The side of the cell that carries the hair-like structures featured many small mitochondria; however, on the opposite side, which is in contact with neurons, the mitochondria formed a single large network. The co-existence of different types of mitochondria within one cell is a novel concept. Further experiments investigated how these mitochondrial characteristics were connected to hair cell activity. They showed that this organisation was established gradually as the cells aged, with cellular activity shaping the architecture (but not the total volume) of the mitochondria. Overall, the work by McQuate et al. provides important information necessary to develop therapeutics for hearing disorders linked to mitochondrial dysfunction. However, by showing that various kind of mitochondria can be present within one cell, it should also inform studies beyond those that focus on hearing.

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.

NALTREXONE AS A PROMISING TREATMENT FOR CLINICAL SIGNS OF LATERAL LINE DEPIGMENTATION IN PALETTE SURGEONFISH (PARACANTHURUS HEPATUS).

Lateral line depigmentation (LLD) is a common condition in managed tropical saltwater fish, and treatment is somewhat elusive. Naltrexone, an opioid receptor antagonist, enhances epithelial cell replication, cytokine production, and angiogenesis to stimulate wound healing in mice. A treatment trial with 11 palette surgeonfish with LLD was performed. Seven fish underwent a single topical treatment of a mixture of 4 mg naltrexone and 10 g iLEX petroleum paste applied topically to LLD lesions. Four additional fish served as controls: two received only topical iLEX and two received no treatment. Severity of disease was scored on a 0-3 scale. Inflammatory response was gauged on a separate 0-3 scale for 5 d after treatment based on severity of erythema, as seen in a clinical case performed prior to this study. After 11 days, four affected animals that lacked an inflammatory response after naltrexone topical treatment were administered a single dose of intralesional 0.04% naltrexone (4 mg diluted into 10 ml saline). Lesions on all fish were photographed and measured at day 33. Clinical improvements in lesion size and pigmentation were apparent following topical naltrexone therapy in fish with severe lesions. Although these cases are promising, more data are needed to further evaluate the effectiveness of naltrexone 0.04% in treating LLD lesions in palette surgeonfish.

Loss of ndrg2 Function Is Involved in Notch Activation in Neuromast Hair Cell Regeneration in Zebrafish.

The regeneration of hair cells in zebrafish is a complex process involving the precise regulation of multiple signaling pathways, but this complicated regulatory network is not fully understood. Current research has primarily focused on finding molecules and pathways that can regulate hair cell regeneration and restore hair cell functions. Here, we show the role of N-Myc downstream regulated gene 2 (ndrg2) in zebrafish hair cell regeneration. We first found that ndrg2 was dynamically expressed in neuromasts of the developing zebrafish, and this expression was increased after neomycin-induced hair cell damage. Then, ndrg2 loss-of-function larvae showed reduced numbers of regenerated hair cells but increased numbers of supporting cells after neomycin exposure. By in situ hybridization, we further observed that ndrg2 loss of function resulted in the activation of Notch signaling and downregulation of atoh1a during hair cell regeneration in vivo. Additionally, blocking Notch signaling rescued the number of regenerated hair cells in ndrg2-deficient larvae. Together, this study provides evidence for the role of ndrg2 in regulating hair cell regeneration in zebrafish neuromasts.

Lateral line ablation by ototoxic compounds results in distinct rheotaxis profiles in larval zebrafish.

The zebrafish lateral line is an established model for hair cell organ damage, yet few studies link mechanistic disruptions to changes in biologically relevant behavior. We used larval zebrafish to determine how damage via ototoxic compounds impact rheotaxis. Larvae were treated with CuSO4 or neomycin to disrupt lateral line function then exposed to water flow stimuli. Their swimming behavior was recorded on video then DeepLabCut and SimBA software were used to track movements and classify rheotaxis behavior, respectively. Lateral line-disrupted fish performed rheotaxis, but they swam greater distances, for shorter durations, and with greater angular variance than controls. Furthermore, spectral decomposition analyses confirmed that lesioned fish exhibited ototoxic compound-specific behavioral profiles with distinct changes in the magnitude, frequency, and cross-correlation between fluctuations in linear and angular movements. Our observations demonstrate that lateral line input is needed for fish to hold their station in flow efficiently and reveals that commonly used lesion methods have unique effects on rheotaxis behavior.