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.

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.

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.

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.

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.

Alone in a crowd: effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2.

Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide for which thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish in which lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially raised lateral line mutants express lower levels of pth2 relative to wild-type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a form of isolation, even when raised in a social environment. This article has an associated First Person interview with the first author of the paper.

Dnmt1 is required for the development of auditory organs via cell cycle arrest and Fgf signalling.

OBJECTIVES: To explore the role of DNA methyltransferase 1 (DNMT1) in the development of auditory system using zebrafish as experimental model. METHODS: Morpholino oligonucleotide was used to induce Dnmt1 deficiency. RNA sequencing, in situ hybridization (ISH), whole genomic bisulfide sequencing (WGBS) and immunostaining were used to investigate the morphologic alterations and mechanisms. RESULTS: We found that downregulation of Dnmt1 induced decreased number of neuromasts and repressed cell proliferation of primordium in the developing posterior lateral line system of zebrafish. The ISH data uncovered that Fgf signalling pathway was inhibited and the expression of chemokine members cxcr4b, cxcr7b and cxcl12a were interfered, while lef1 expression was increased after inhibiting Dnmt1. Additionally, Dnmt1 downregulation led to malformed otoliths and deformed semicircular canals, and hair cell differentiation in utricle and saccule was inhibited severely. The in situ staining of otic placode markers pax2/5 and fgf 3/8/10 was decreased when Dnmt1 downregulated. The WGBS analysis demonstrated that the global methylation status was markedly downregulated, and cell cycle genes were among those most differently expressed between Dnmt1 morphants and the controls. Further ISH analysis confirmed the findings by RNA-seq and WGBS assay that cdkn1a and tp53 were both upregulated after knockdown of Dnmt1. CONCLUSION: Our results revealed that Dnmt1 is essential for the development of zebrafish auditory organ through regulating cell cycle genes together with Wnt and Fgf signalling pathways.

An enhancer trap zebrafish line for lateral line development and regulation of six2b expression.

Zebrafish lateral line system which is derived from neurogenic placodes has become a popular model for developmental biology since its formation involves cell migration, pattern formation, organogenesis, and hair cell regeneration. Transgenic lines play a crucial role in lateral line system study. Here, we identified an enhancer trap transgenic zebrafish line Et(gata2a:EGFP)189b (ET189b for short), which expressed enhanced green fluorescent protein (EGFP) in the pituitary, otic, and lateral line placodes and their derivatives. Especially, in neuromast, the accessory cells rather than hair cells were labeled by EGFP. Furthermore, we found the Tol2 transposon construct is integrated at the proximal upstream region of six2b gene locus. And EGFP expression of ET189b closely reflects the expression of endogenous six2b during development and after dkk1b over-expression. Taken together, our results indicated that ET189b is an ideal line for research on lateral line development and regulation of six2b expression.

Localization of Neurotrophin Specific Trk Receptors in Mechanosensory Systems of Killifish (Nothobranchius guentheri).

Neurotrophins (NTs) and their signal-transducing Trk receptors play a crucial role in the development and maintenance of specific neuronal subpopulations in nervous and sensory systems. NTs are supposed to regulate two sensory systems in fish, the inner ear and the lateral line system (LLS). The latter is one of the major mechanosensory systems in fish. Considering that annual fishes of the genus Nothobranchius, with their short life expectancy, have become a suitable model for aging studies and that the occurrence and distribution of neurotrophin Trk receptors have never been investigated in the inner ear and LLS of killifish (Nothobranchius guentheri), our study aimed to investigate the localization of neurotrophin-specific Trk receptors in mechanosensory systems of N. guentheri. For histological and immunohistochemical analysis, adult specimens of N. guentheri were processed using antibodies against Trk receptors and S100 protein. An intense immunoreaction for TrkA and TrkC was found in the sensory cells of the inner ear as well as in the hair cells of LLS. Moreover, also the neurons localized in the acoustic ganglia displayed a specific immunoreaction for all Trk receptors (TrkA, B, and C) analyzed. Taken together, our results demonstrate, for the first time, that neurotrophins and their specific receptors could play a pivotal role in the biology of the sensory cells of the inner ear and LLS of N. guentheri and might also be involved in the hair cells regeneration process in normal and aged conditions.

Notch signaling induces either apoptosis or cell fate change in multiciliated cells during mucociliary tissue remodeling.

Multiciliated cells (MCCs) are extremely highly differentiated, presenting >100 cilia and basal bodies. Therefore, MCC fate is thought to be terminal and irreversible. We analyzed how MCCs are removed from the airway-like mucociliary Xenopus epidermis during developmental tissue remodeling. We found that a subset of MCCs undergoes lateral line-induced apoptosis, but that the majority coordinately trans-differentiate into goblet secretory cells. Both processes are dependent on Notch signaling, while the cellular response to Notch is modulated by Jak/STAT, thyroid hormone, and mTOR signaling. At the cellular level, trans-differentiation is executed through the loss of ciliary gene expression, including foxj1 and pcm1, altered proteostasis, cilia retraction, basal body elimination, as well as the initiation of mucus production and secretion. Our work describes two modes for MCC loss during vertebrate development, the signaling regulation of these processes, and demonstrates that even cells with extreme differentiation features can undergo direct fate conversion.

Use of zebrafish larvae lateral line to study protection against cisplatin-induced ototoxicity: A scoping review.

AIM: The present review aimed to consolidate and analyze the recent information about the use of zebrafish in studies concerning cisplatin-induced ototoxicity and otoprotection. MATERIAL AND METHODS: The PubMed, Web of Science, and Scopus databanks were searched using the following MESH terms: zebrafish, cisplatin, ototoxicity. The identified publications were screened according to inclusion and exclusion criteria and the 26 qualifying manuscripts were included in the full-text analysis. The experimental protocols, including cisplatin concentrations, the exposure duration and the outcome measurements used in zebrafish larvae studies, were evaluated and the reported knowledge was summarized. RESULTS: Twenty-six substances protecting from cisplatin-induced toxicity were identified with the use of zebrafish larvae. These substances include quinine, salvianolic acid B, berbamine 6, benzamil, quercetin, dexmedetomidine, dexamethsanone, quinoxaline, edaravone, apocynin, dimethyl sulfoxide, KR-22335, SRT1720, ORC-13661, 3-MA, D-methionine, mdivi-1, FUT-175, rapamycin, Z-LLF-CHO, ATX, NAC, CYM-5478, CHCP1, CHCP2 and leupeptin. The otoprotective effects of compounds were attributed to their anti-ROS, anti-apoptotic and cisplatin uptake-blocking properties. The broadest range of protection was achieved when the experimental flow used preconditioning with an otoprotective compound and later a co-incubation with cisplatin. Protection against a high concentration of cisplatin was observed only in protocols using short exposure times (4 and 6 h). CONCLUSIONS: The data extracted from the selected papers confirm that despite the differences between the human and the zebra fish hearing thresholds (as affected by cisplatin), the sensory cells of zebrafish and larval zebrafish are a valuable tool which could be used: (i) for the discovery of novel otoprotective substances and compounds; (ii) to screen their side effects and (iii) to extend the knowledge on the mechanisms of cisplatin-induced inner ear damage. For future studies, the development of a consensus experimental protocol is highly recommended.

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development.

Transcription factors that contain a homeodomain DNA-binding domain have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a subfamily of NK homeodomain-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here, we show that zebrafish (Danio rerio) hmx2 and hmx3a are coexpressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1, and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development.

The expression of zebrafish NAD(P)H:quinone oxidoreductase 1(nqo1) in adult organs and embryos.

NQO1, NAD(P)H: quinone oxidoreductase 1, was first identified in rat and its role has been extensively studied. Even the roles of NQO1 in the maintenance of physiological function and disease were largely addressed, whether the tissue specific functions of the NQO1 in organ development remains unknown. In the current study, we identified two NQO1 isoforms (isoform 1 and isoform 2) and examined the expression of nqo1 variants in adult zebrafish organs and embryos at different stages. In adult organs, RT-PCR result indicated that nqo1 variant 1 was mainly expressed in stomach and intestine, while nqo1 variant 2 was expressed in all organs investigated except for heart. Further, RT-PCR result showed that the nqo1 variant 1 and variant 2 were expressed at all the embryonic stages, but nqo1 variant 1 expression level was much lower than that of nqo1 variant 2. To specifically examine the expression pattern of these two different nqo1 variants, we did whole mount in situ hybridization and the results demonstrated that, both of them were maternally expressed at 8-cell stage, and they were all expressed ubiquitously at early stage. At 24 hpf, nqo1 variant 2 was mainly expressed in yolk cells, and slightly in head and eyes. At 48 hpf, nqo1 variant 2 was restricted in lateral line neuromasts. From 72 hpf to 144 hpf, nqo1 variant 2 was mainly restricted in branchial arch, liver, swimming bladder and lateral line neuromasts, while from 124 hpf to 192 hpf, nqo1 variant 2 only restricted in liver, and disappeared in lateral line neuromasts. On the contrary, at the late embryonic stage, nqo1 variant 1 was only expressed in liver and swimming bladder while not in branchial arch and lateral line neuromasts. In conclusion, we systematically analyzed the expression pattern of nqo1 variant 1 and variant 2 in zebrafish at different embryonic stages, and our data implied the possible role of nqo1 in regulating liver, branchial arch and lateral neuromasts development.

Yap-lin28a axis targets let7-Wnt pathway to restore progenitors for initiating regeneration.

The sox2 expressing (sox2+) progenitors in adult mammalian inner ear lose the capacity to regenerate while progenitors in the zebrafish lateral line are able to proliferate and regenerate damaged HCs throughout lifetime. To mimic the HC damage in mammals, we have established a zebrafish severe injury model to eliminate both progenitors and HCs. The atoh1a expressing (atoh1a+) HC precursors were the main population that survived post severe injury, and gained sox2 expression to initiate progenitor regeneration. In response to severe injury, yap was activated to upregulate lin28a transcription. Severe-injury-induced progenitor regeneration was disabled in lin28a or yap mutants. In contrary, overexpression of lin28a initiated the recovery of sox2+ progenitors. Mechanistically, microRNA let7 acted downstream of lin28a to activate Wnt pathway for promoting regeneration. Our findings that lin28a is necessary and sufficient to regenerate the exhausted sox2+ progenitors shed light on restoration of progenitors to initiate HC regeneration in mammals.

Characterization of a nap1l1 transgenic reporter in zebrafish.

Nap1l1 gene encodes a tissue specific nucleosome assembly protein and is essential for tissue development. Here, we report the generation and characterization of a nap1l1 transgenic reporter in zebrafish model. We showed that a 5-kilobase (kb) genomic fragment immediately upstream of the nap1l1 gene transcription initiation site is capable of targeting the nucleic enhanced green fluorescence protein (EGFP) expression initially to central nervous system and subsequently to lateral line neuromasts, cardiomyocytes, and paraxial vessels, where the endogenous nap1l1 normally expresses with only a few exception. In adulthood, zebrafish nap1l1 promoter-driving nEGFP is predominantly expressed in lateral line system, liver, and ovary, but not in heart. Therefore, this novel transgenic reporter line, Tg(nap1l1:nEGFP)zs102, would be a valuable tool for studying the development and regeneration of lateral line system and also for investigating cardiac development.

Characterization of biklf/klf17-deficient zebrafish in posterior lateral line neuromast and hatching gland development.

Krüpple-like factors (Klfs) are highly conserved zinc-finger transcription factors that regulate various developmental processes, such as haematopoiesis and cardiovascular development. In zebrafish, transient knockdown analysis of biklf/klf17 using antisense morpholino suggests the involvement of biklf/klf17 in primitive erythropoiesis and hatching gland development; however, the continuous physiological importance of klf17 remains uncharacterized under the genetic ablation of the klf17 gene among vertebrates. We established the klf17-disrupted zebrafish lines using the CRISPR/Cas9 technology and performed phenotypic analysis throughout early embryogenesis. We found that the klf17-deficient embryos exhibited abnormal lateral line neuromast deposition, whereas the production of primitive erythrocytes and haemoglobin production were observed in the klf17-deficient embryos. The expression of lateral line neuromast genes, klf17 and s100t, in the klf17-deficient embryos was detected in posterior lateral line neuromasts abnormally positioned at short intervals. Furthermore, the klf17-deficient embryos failed to hatch and died without hatching around 15 days post-fertilization (dpf), whereas the dechorionated klf17-deficient embryos and wild-type embryos were alive at 15 dpf. The klf17-deficient embryos abolished hatching gland cells and Ctsl1b protein expression, and eliminated the expression of polster and hatching gland marker genes, he1.1, ctsl1b and cd63. Thus, the klf17 gene plays important roles in posterior lateral line neuromast and hatching gland development.

Shootins mediate collective cell migration and organogenesis of the zebrafish posterior lateral line system.

The zebrafish sensory posterior lateral line is an excellent model system to study collective cell migration and organogenesis. Shootin1 is a cytoplasmic protein involved in neuronal polarization and axon guidance. Previous studies have shown that shootin1 couples actin filament retrograde flow with extracellular adhesive substrates at the leading edge of axonal growth cones, thereby producing mechanical force for the migration and guidance of axonal growth cones. However, the functions of shootin in peripheral cells remain unknown. Here we identified two novel shootin family members, shootin2 and shootin3. In zebrafish, shootin1 and shootin3 are expressed in the posterior lateral line primordium (PLLP) and neuromasts during embryonic development. A shootin1 mutant displayed a reduced speed of PLLP migration, while shootin1;shootin3 double mutation inhibited cell proliferation in the PLLP. Furthermore, our results suggest that shootin1 and shootin3 positively regulate the number of neuromasts and the number of cells in deposited neuromasts. Our study demonstrates that shootins mediate collective cell migration of the posterior lateral line primordium and formation of neuromasts in zebrafish.

Differences in Sodium Channel Densities in the Apical Dendrites of Pyramidal Cells of the Electrosensory Lateral Line Lobe.

Heterogeneity of neural properties within a given neural class is ubiquitous in the nervous system and permits different sub-classes of neurons to specialize for specific purposes. This principle has been thoroughly investigated in the hindbrain of the weakly electric fish A. leptorhynchus in the primary electrosensory area, the Electrosensory Lateral Line lobe (ELL). The pyramidal cells (PCs) that receive inputs from tuberous electroreceptors are organized in three maps in distinct segments of the ELL. The properties of these cells vary greatly across maps due to differences in connectivity, receptor expression, and ion channel composition. These cells are a seminal example of bursting neurons and their bursting dynamic relies on the presence of voltage-gated Na+ channels in the extensive apical dendrites of the superficial PCs. Other ion channels can affect burst generation and their expression varies across ELL neurons and segments. For example, SK channels cause hyperpolarizing after-potentials decreasing the likelihood of bursting, yet bursting propensity is similar across segments. We question whether the depolarizing mechanism that generates the bursts presents quantitative differences across segments that could counterbalance other differences having the opposite effect. Although their presence and role are established, the distribution and density of the apical dendrites' Na+ channels have not been quantified and compared across ELL maps. Therefore, we test the hypothesis that Na+ channel density varies across segment by quantifying their distribution in the apical dendrites of immunolabeled ELL sections. We found the Na+ channels to be two-fold denser in the lateral segment (LS) than in the centro-medial segment (CMS), the centro-lateral segment (CLS) being intermediate. Our results imply that this differential expression of voltage-gated Na+ channels could counterbalance or interact with other aspects of neuronal physiology that vary across segments (e.g., SK channels). We argue that burst coding of sensory signals, and the way the network regulates bursting, should be influenced by these variations in Na+ channel density.

Wnt/ß-catenin interacts with the FGF pathway to promote proliferation and regenerative cell proliferation in the zebrafish lateral line neuromast.

Wnt and FGF are highly conserved signaling pathways found in various organs and have been identified as important regulators of auditory organ development. In this study, we used the zebrafish lateral line system to study the cooperative roles of the Wnt and FGF pathways in regulating progenitor cell proliferation and regenerative cell proliferation. We found that activation of Wnt signaling induced cell proliferation and increased the number of hair cells in both developing and regenerating neuromasts. We further demonstrated that FGF signaling was critically involved in Wnt-regulated proliferation, and inhibition of FGF abolished the Wnt stimulation-mediated effects on cell proliferation, while activating FGF signaling with basic fibroblast growth factor (bFGF) led to a partial rescue of the proliferative failure and hair cell defects in the absence of Wnt activity. Whole-mount in situ hybridization analysis showed that the expression of several FGF pathway genes, including pea3 and fgfr1, was increased in neuromasts after treatment with the Wnt pathway inducer BIO. Interestingly, when SU5402 was used to inhibit FGF signaling, neuromast cells expressed much lower levels of the FGF receptor gene, fgfr1, but produced increased levels of Wnt target genes, including ctnnb1, ctnnb2, and tcf7l2, while bFGF treatment produced no alterations in the expression of those genes, suggesting that fgfr1 might restrict Wnt signaling in neuromasts during proliferation. In summary, our analysis demonstrates that both the Wnt and FGF pathways are tightly integrated to modulate the proliferation of progenitor cells during early neuromast development and regenerative cell proliferation after neomycin-induced injury in the zebrafish neuromast.

Glucococorticoid receptor activation exacerbates aminoglycoside-induced damage to the zebrafish lateral line.

Aminoglycoside antibiotics have potent antibacterial properties but cause hearing loss in up to 25% of patients. These drugs are commonly administered in patients with high glucocorticoid stress hormone levels and can be combined with exogenous glucocorticoid treatment. However, the interaction of stress and aminoglycoside-induced hearing loss has not been fully explored. In this study, we investigated the effect of the glucocorticoid stress hormone cortisol on hair cells in the zebrafish lateral line as an important step toward understanding how physiological stressors modulate hair cell survival. We found that 24-hr cortisol incubation sensitized hair cells to neomycin damage. Pharmacological and genetic manipulation demonstrates that sensitization depended on the action of the glucocorticoid receptor but not the mineralocorticoid receptor. Blocking endogenous cortisol production reduced hair cell susceptibility to neomycin, further evidence that glucocorticoids modulate aminoglycoside ototoxicity. Glucocorticoid transcriptional activity was apparent in lateral line hair cells, suggesting a direct action of cortisol in these aminoglycoside-sensitive cells. Our work shows that the stress hormone cortisol can increase hair cell sensitivity to aminoglycoside damage, which highlights the importance of recognizing stress and the impacts of glucocorticoid signaling in both ototoxicity research and clinical practice.