Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish.

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Development of the cranial lateral line system of Brook Trout, Salvelinus fontinalis (Teleostei: Salmonidae): Evolutionary and ecological implications.

The mechanosensory lateral line (LL) system of salmonid fishes has been the focus of comparative morphological studies and behavioral and physiological analyses of flow sensing capabilities, but its morphology and development have not been studied in detail in any one species. Here, we describe the post-embryonic development of the cranial LL system in Brook Trout, Salvelinus fontinalis, using vital fluorescent staining (4-Di-2-ASP), scanning electron microscopy, µCT, and clearing and staining to visualize neuromasts and the process of cranial LL canal morphogenesis. We examined the relationship between the timing of LL development, the prolonged life history of salmonids, and potential ecological implications. The LL system is composed of seven canals containing canal neuromasts (CNs) and four lines of superficial neuromasts (SNs) on the skin. CNs and SNs increase in number and size during the alevin (larval) stage. CN number stabilizes as canal morphogenesis commences, but SN number increases well into the parr (juvenile) stage. CNs become larger and more elongated than SNs, but the relative area occupied by sensory hair cells decreases during ontogeny in both types of neuromasts. Neuromast-centered canal morphogenesis starts in alevins (yolk sac larvae), as they swim up into the water column from their gravel nests (~4 months post-fertilization), after which yolk sac absorption is completed and exogenous feeding begins. Canal morphogenesis proceeds asynchronously within and among canal series and is not complete until ~8 months post-fertilization (the parr stage). Three characters in the LL system and associated dermal bones were used to identify their homologs in other actinopterygians and to consider the evolution of LL canal reduction, thus demonstrating the value of salmonids for the study of LL evolution. The prolonged life history of Brook Trout and the onset of canal morphogenesis at swim-up are predicted to have implications for neuromast function at these critical behavioral and ecological transitions.

Pharmacological reprogramming of zebrafish lateral line supporting cells to a migratory progenitor state.

In the zebrafish lateral line, non-sensory supporting cells readily re-enter the cell cycle to generate new hair cells and supporting cells during homeostatic maintenance and following damage to hair cells. This contrasts with supporting cells from mammalian vestibular and auditory sensory epithelia which rarely re-enter the cell cycle, and hence loss of hair cells results in permanent sensory deficit. Lateral line supporting cells are derived from multipotent progenitor cells that migrate down the trunk midline as a primordium and are deposited to differentiate into a neuromast. We have found that we can revert zebrafish support cells back to a migratory progenitor state by pharmacologically altering the signaling environment to mimic that of the migratory primordium, with active Wnt signaling and repressed FGF signaling. The reverted supporting cells migrate anteriorly and posteriorly along the horizontal myoseptum and will re-epithelialize to form an increased number of neuromasts along the midline when the pharmacological agents are removed. These data demonstrate that supporting cells can be readily reprogrammed to a migratory multipotent progenitor state that can form new sensory neuromasts, which has important implications for our understanding of how the lateral line system matures and expands in fish and also suggest avenues for returning mammalian supporting cells back to a proliferative state.

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.

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorate aminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5 days' post fertilization after neomycin (20 µM) and gentamicin (20 mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100 µM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300 µM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20 µM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake of neomycin into hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production and aminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

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.

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.

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.

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.

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.

Reinterpreting the work of Charles Breder: Sensory neuromasts and orbital skeleton variation in eyeless Astyanax cavefish.

Charles Breder, a pioneering researcher of blind Mexican cavefish was the first to note extreme variation in the facial skeleton of this intriguing subterranean-dwelling organism. Using a system of polar coordinate plots, he identified substantial dysmorphic changes affecting bones of the orbital skeleton. A complication of his landmark publication from 1944 was an error in the number of orbital bones depicted for this species. Intriguingly, however, he proposed an unknown "organizing force" likely influences final bone position and associated dysmorphia. At the time this was merely hypothetical. Roughly eight decades since its publication, however, insights into sensory influences on facial bone development may explain dysmorphia and variation in bone numbers for Astyanax cavefish. A morphological association between mechano-sensory neuromasts of the lateral line and dermal bones of the facial skeleton had been appreciated in the classical literature, but the polarity of this interaction has long remained unclear. Here, we propose that sensory-skeletal integration between sensory neuromasts and bones explain the incomplete numbers of bones, and dysmorphic features such as fusion between neighboring elements. We propose that in closely-related surface fish (and most teleost fish) this developmental coupling enables the sensory and skeletal systems to become integrated into a functional unit over the course of life history. In this opinion article, we discuss the relevance of this (poorly understood) phenomenon as a potential evolutionary source of variation in the facial bone structures of taxa across deep geologic time. We provide three potential explanations for the error in Breder's drawings, that may be explained by natural developmental variation documented in other related species. Moreover, we argue that the natural variation in this "evolutionary" model system is useful for explaining diverse cranial features by uniting aberrations occurring during embryogenesis with long-term adult dysmorphia.

The H3K27 demethylase controls the lateral line embryogenesis of zebrafish.

BACKGROUND: Kdm6b, a specific histone 3 lysine 27 (H3K27) demethylase, has been reported to be implicated in a variety of developmental processes including cell differentiation and cell fate determination and multiple organogenesis. Here, we regulated the transcript level of kdm6bb to study the potential role in controlling the hearing organ development of zebrafish. METHODS: A morpholino antisense oligonucleotide (MO) strategy was used to induce Kdm6b deficiency; immunohistochemical staining and in situ hybridization analysis were conducted to figure out the morphologic alterations and embryonic mechanisms. RESULTS: Kdm6bb is expressed in the primordium and neuromasts at the early stage of zebrafish embryogenesis, suggesting a potential function of Kdm6b in the development of mechanosensory organs. Knockdown of kdm6bb severely influences the cell migration and proliferation in posterior lateral line primordium, abates the number of neuromasts along the trunk, and mRNA-mediated rescue test can partially renew the neuromasts. Loss of kdm6bb might be related to aberrant expressions of chemokine genes encompassing cxcl12a and cxcr4b/cxcr7b in the migrating primordium. Moreover, inhibition of kdm6bb reduces the expression of genes in Fgf signaling pathway, while it increases the axin2 and lef1 expression level of Wnt/ß-catenin signaling during the migrating stage. CONCLUSIONS: Collectively, our results revealed that Kdm6b plays an essential role in guiding the migration of primordium and in regulating the deposition of zebrafish neuromasts by mediating the gene expression of chemokines and Wnt and Fgf signaling pathway. Since histone methylation and demethylation are reversible, targeting Kdm6b may present as a novel therapeutic regimen for hearing disorders.

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.

Evaluation of Cisplatin-Induced Pathology in the Larval Zebrafish Lateral Line.

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells-the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM-1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4-8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.

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.