Regeneration in zebrafish lateral line neuromasts: expression of the neural progenitor cell marker sox2 and proliferation-dependent and-independent mechanisms of hair cell renewal

Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.     

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.     

Rheotaxis in larval zebrafish is mediated by lateral line mechanosensory hair cells

The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration.     

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.¹⁷ that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.     

atp2b1a regulates Ca(2+) export during differentiation and regeneration of mechanosensory hair cells in zebrafish

The molecular mechanisms of development of mechanosensory hair cells have been tackled successfully due to in vivo studies in the zebrafish lateral line. The enhancer trap (ET) transgenic line, SqET4 was instrumental in these studies even despite a lack of a link of its GFP expression pattern to a particular gene(s). We mapped the Tol2 transposon insertion of the SqET4 transgenics onto Chr. 4 next to a gene encoding Atp2b1a (Pmca1) - one of the four PMCAs acting to export Ca(2+) from a cell. atp2b1a expression recapitulates that of GFP during the development of mechanoreceptors of the inner ear and lateral line. atp2b1a expression correlates with the regeneration of these cells. Thus, SqET4 represents the Tg:atp2b1a-GFP line, which links Ca(2+) metabolism and the differentiation of mechanoreceptors. The morpholino-mediated knockdown of atp2b1a blocks Ca(2+) export and affects the division of hair cell progenitors, resulting in their accumulation. Under the control of a master gene of hair cells, Atoh1a, Atp2b1a functions during progenitor cell proliferation and hair cell differentiation. Given the similarity between the phenotypes of atp2b1a morphants and embryos treated with the pan-PMCA inhibitor 5(6)-carboxyeosin, Atp2b1a emerges as member of the Atp2b family responsible for Ca(2+) export during the development of hair cells.     

Compartmentalized Notch signaling sustains epithelial mirror symmetry

Bilateral symmetric tissues must interpret axial references to maintain their global architecture during growth or repair. The regeneration of hair cells in the zebrafish lateral line, for example, forms a vertical midline that bisects the neuromast epithelium into perfect mirror-symmetric plane-polarized halves. Each half contains hair cells of identical planar orientation but opposite to that of the confronting half. The establishment of bilateral symmetry in this organ is poorly understood. Here, we show that hair-cell regeneration is strongly directional along an axis perpendicular to that of epithelial planar polarity. We demonstrate compartmentalized Notch signaling in neuromasts, and show that directional regeneration depends on the development of hair-cell progenitors in polar compartments that have low Notch activity. High-resolution live cell tracking reveals a novel process of planar cell inversions whereby sibling hair cells invert positions immediately after progenitor cytokinesis, demonstrating that oriented progenitor divisions are dispensable for bilateral symmetry. Notwithstanding the invariably directional regeneration, the planar polarization of the epithelium eventually propagates symmetrically because mature hair cells move away from the midline towards the periphery of the neuromast. We conclude that a strongly anisotropic regeneration process that relies on the dynamic stabilization of progenitor identity in permissive polar compartments sustains bilateral symmetry in the lateral line.     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014     

Profiling drug-induced cell death pathways in the zebrafish lateral line

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while d-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.     

Ethanol Affects the Development of Sensory Hair Cells in Larval Zebrafish (Danio rerio)

Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS). The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf) and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%–1.75% by volume) from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.     

Epicatechin protects auditory cells against cisplatin-induced death

Cisplatin, a chemotherapeutic drug that is widely used to treat various cancers, promotes ototoxicity at higher doses. In this study, the effect of epicatechin (EC) on cisplatin-induced hair cell death was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1, and in vivo in zebrafish. Cisplatin promoted apoptosis and altered mitochondrial membrane potential (MMP) in HEI-OC1 cells. EC inhibited cisplatin-induced apoptosis and intracellular reactive oxygen species (ROS) generation. Labeling of zebrafish lateral line hair cells by the fluorescent dye YO-PRO1 was lost upon exposure to cisplatin, and EC protected against this cisplatin-induced loss of labeling in a dose-dependent manner. Scanning and transmission electron micrographs showed that treatment with EC protected against cisplatin-induced loss of kinocilium and stereocilia in zebrafish neuromasts. These results suggest that EC prevents cisplatin-induced ototoxicity by blocking ROS generation and by preventing changes in MMP.     

Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina

Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones.     

Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

Growth hormone promotes hair cell regeneration in the zebrafish (Danio rerio) inner ear following acoustic trauma

Background

Previous microarray analysis showed that growth hormone (GH) was significantly upregulated following acoustic trauma in the zebrafish (Danio rerio) ear suggesting that GH may play an important role in the process of auditory hair cell regeneration. Our objective was to examine the effects of exogenous and endogenous GH on zebrafish inner ear epithelia following acoustic trauma.

Methodology/Principal Findings

We induced auditory hair cell damage by exposing zebrafish to acoustic overstimulation. Fish were then injected intraperitoneally with either carp GH or buffer, and placed in a recovery tank for either one or two days. Phalloidin-, bromodeoxyuridine (BrdU)-, and TUNEL-labeling were used to examine hair cell densities, cell proliferation, and apoptosis, respectively. Two days post-trauma, saccular hair cell densities in GH-treated fish were similar to that of baseline controls, whereas buffer-injected fish showed significantly reduced densities of hair cell bundles. Cell proliferation was greater and apoptosis reduced in the saccules, lagenae, and utricles of GH-treated fish one day following trauma compared to controls. Fluorescent in situ hybridization (FISH) was used to examine the localization of GH mRNA in the zebrafish ear. At one day post-trauma, GH mRNA expression appeared to be localized perinuclearly around erythrocytes in the blood vessels of the inner ear epithelia. In order to examine the effects of endogenous GH on the process of cell proliferation in the ear, a GH antagonist was injected into zebrafish immediately following acoustic trauma, resulting in significantly decreased cell proliferation one day post-trauma in all three zebrafish inner ear end organs.

Conclusions/Significance

Our results show that exogenous GH promotes post-trauma auditory hair cell regeneration in the zebrafish ear through stimulating proliferation and suppressing apoptosis, and that endogenous GH signals are present in the zebrafish ear during the process of auditory hair cell regeneration.     

Melanocyte regeneration reveals mechanisms of adult stem cell regulation

Utilization of adult stem cells in regenerative therapies may require a thorough understanding of the mechanisms that establish, recruit and renew the stem cell, promote the differentiation of its daughters, or how the stem cell is repressed by its target tissue. Regeneration of melanocytes in the regenerating zebrafish caudal fin, or following larval melanocyte-specific ablation, or recruitment of new melanocytes during pigment pattern metamorphosis each provides evidence for melanocyte stem cells (MSCs) that support the melanocyte pigment pattern. We discuss the mechanisms of MSC regulation provided from analysis of normal or mutant regeneration in each of these systems, including the implications drawn from evidence that regeneration does not simply recapitulate ontogenetic development. These results suggest that analysis of melanocyte regeneration in zebrafish will provide a fine scale dissection of mechanisms establishing or regulating adult stem cells.     

Expression of proneural and neurogenic genes in the zebrafish lateral line primordium correlates with selection of hair cell fate in neuromasts

Expression of a mouse atonal homologue, math1, defines cells with the potential to become sensory hair cells in the mouse inner ear (Science 284 (1999) 1837) and Notch signaling limits the number of cells that are permitted to adopt this fate (Nat. Genet. 21 (1999) 289; J. Neurocytol. 28 (1999) 809). Failure of lateral inhibition mediated by Notch signaling is associated with an overproduction of ear hair cells in the zebrafish mind bomb (mib) and deltaA mutants (Development 125 (1998a) 4637; Development 126 (1999) 5669), suggesting a similar role for these genes in limiting the number of hair cells in the zebrafish ear. This study extends the analysis of proneural and neurogenic gene expression to the lateral line system, which detects movement via clusters of related sensory hair cells in specialized structures called neuromasts. We have compared the expression of a zebrafish atonal homologue, zath1, and neurogenic genes, deltaA, deltaB and notch3, in neuromasts and the posterior lateral line primordium (PLLP) of wild-type and mib mutant embryos. We describe progressive restriction of proneural and neurogenic gene expression in the migrating PLLP that appears to correlate with selection of hair cell fate in maturing neuromasts. In mib mutants there is a failure to restrict expression of zath1 and Delta homologues in the neuromasts revealing similarities with the phenotype previously described in the ear.     

Defective calmodulin‐dependent rapid apical endocytosis in zebrafish sensory hair cell mutants

Vertebrate mechanosensory hair cells contain a narrow “pericuticular” zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1‐43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 424–434, 1999     

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Coordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF(4T)(Δ)(2B) promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.     

Regulation of latent sensory hair cell precursors by glia in the zebrafish lateral line

The lateral line is a placodally derived mechanosensory organ in anamniotes that detects the movement of water. In zebrafish embryos, a migrating primordium deposits seven to nine clusters of sensory hair cells, or neuromasts, at intervals along the trunk. Postembryonically, neuromasts continue to be added. We show that some secondary neuromasts arise from a pool of latent precursors that are deposited by the primordium between primary neuromasts. Interneuromast cells lie adjacent to the lateral line nerve and associated glia. These cells remain quiescent while they are juxtaposed with the glia; however, when they move away from the nerve they increase proliferation and form neuromasts. If glia are manually removed or genetically ablated by mutations in cls/sox10, hypersensitive (hps), or rowgain (rog), neuromasts precociously differentiate. Transplantation of wt glia into mutants rescues the appropriate temporal differentiation of interneuromast cells. Our studies reveal a role for glia in regulating sensory hair cell precursors.