Short Stereocilia Research Articles

Voltage-dependent bundle motion in mammalian auditory hair cells

Auditory hair cells have bundles of protrusions on their surface held together by proteinaceous links. Sound-induced vibrations deflect these bundles, transferring force through the links to open nearby mechanically sensitive ion channels and providing our sensation of sound. An increase in bundle compliance caused by channel gating can also be detected as motion. The channels open during depolarization and the bundle moves rapidly toward shorter stereocilia, followed by a larger but slower movement toward the taller stereocilia. To determine how voltage changes affect mammalian inner hair cell (IHC) bundles, we used whole cell patch clamp and high-speed imaging to apply electrical stimulation and measure bundle motion. We found two components in the motions of freestanding IHC bundles in response to depolarization, a fast offset away from the tallest row and a slower movement back toward the tallest row. Like in turtles, the slower motion was sensitive to channel block and suggested ion flux, particularly calcium, played a role. Our findings show IHCs can rapidly respond to changes in the bundle setpoint without ion influx. This work improves our understanding of the mechano-electrical transduction process and helps address the controversial role of calcium in regulating the channel current response.

Hair cell morphological patterns and polarity organization in the sea lamprey vestibular cristae.

The inner ear of the sea lamprey was examined by scanning electron microscopy, antibody labeling with tubulin, Myo7a, Spectrin, and Phalloidin stain to elucidate the canal cristae organization and the morphology and polarity of the hair cells. We characterized the hair cell stereocilia bundles and their morphological polarity with respect to the kinocilia. We identified three types of hair cells. In Type 1 hair cells, the kinocilia were slightly longer than the tallest stereocilia. This type was located along the medial bank of the crista and their polarity, based on kinocilia location, was uniformly pointed ampullipetally. Type 2 hair cells that had kinocilia that were much longer than the stereocilia, were most abundant in the central region of the crista. This type of hair cell displayed variable polarity. Type 3 hair cells had extremely long kinocilia (~40-50 μm long) and with extremely short stereocilia. They were mostly located in the lateral zone crista and displayed ampullipetal polarity. Myo7a and tubulin antibodies revealed that hair cells and vestibular afferents are distributed across the canal cristae in the lamprey, covering the area of cruciate eminence; a feature that is absent in more derived vertebrates. Spectrin shows hair cells of varying polarities in the central zone. In this zone, some cells followed the main polarity vector (lateral) like those in medial and lateral zones, whereas other cells displayed polarities that carried up to 40° from the main polarity vector.

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization.

Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.

Interaction of protocadherin-15 with the scaffold protein whirlin supports its anchoring of hair-bundle lateral links in cochlear hair cells

The hair bundle of cochlear hair cells is the site of auditory mechanoelectrical transduction. It is formed by three rows of stiff microvilli-like protrusions of graduated heights, the short, middle-sized, and tall stereocilia. In developing and mature sensory hair cells, stereocilia are connected to each other by various types of fibrous links. Two unconventional cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), form the tip-links, whose tension gates the hair cell mechanoelectrical transduction channels. These proteins also form transient lateral links connecting neighboring stereocilia during hair bundle morphogenesis. The proteins involved in anchoring these diverse links to the stereocilia dense actin cytoskeleton remain largely unknown. We show that the long isoform of whirlin (L-whirlin), a PDZ domain-containing submembrane scaffold protein, is present at the tips of the tall stereocilia in mature hair cells, together with PCDH15 isoforms CD1 and CD2; L-whirlin localization to the ankle-link region in developing hair bundles moreover depends on the presence of PCDH15-CD1 also localizing there. We further demonstrate that L-whirlin binds to PCDH15 and CDH23 with moderate-to-high affinities in vitro. From these results, we suggest that L-whirlin is part of the molecular complexes bridging PCDH15-, and possibly CDH23-containing lateral links to the cytoskeleton in immature and mature stereocilia.

Ultrastructural localization of the likely mechanoelectrical transduction channel protein, transmembrane-like channel 1 (TMC1) during development of cochlear hair cells

Transmembrane channel like protein 1 (TMC1) is likely to be a pore-forming subunit of the transduction channel of cochlear hair cells that is mechanically gated by tension on tip links in the stereocilia bundle. To localise TMC1 precisely, we labelled mice cochleae of different ages using custom-made polyclonal antibodies to TMC1 for light and transmission electron microscopy (TEM). Immunofluorescence revealed stereocilia labelling at P9 but not at P3 in apical hair cells. Immunogold labelling for TEM confirmed that labelling was absent at P3, and showed weak labelling at P6 with no stereocilia tip labelling, increasing at P9, with specific tip labelling on shorter stereocilia and some throughout the bundle. At P12 and P21, labelling was refined mostly to stereocilia tips. Quantification showed that labelling overall reached maximum by P12, labelling per tip was relatively constant from P9 to P21, but percent tips labelled was reduced from 16% to 8%. Tmc1−/− showed no labelling. Thus TMC1 occurs at the lower end of the tip link, supporting its presence in the MET complex and likely the channel. Tip localisation from P9 onwards coincides with lipoma HMGIC fusion partner-like 5 (LHFPL5), a protein that may be involved in acquiring/maintaining TMC1 localisation.

Auditory Hair Cell Stereocilia: Balancing Stability to Gain Sensitivity?

Auditory Hair Cell Stereocilia: Balancing Stability to Gain Sensitivity?

Distinct roles of Eps8 in the maturation of cochlear and vestibular hair cells

Several genetic mutations affecting the development and function of mammalian hair cells have been shown to cause deafness but not vestibular defects, most likely because vestibular deficits are sometimes centrally compensated. The study of hair cell physiology is thus a powerful direct approach to ascertain the functional status of the vestibular end organs. Deletion of Epidermal growth factor receptor pathway substrate 8 (Eps8), a gene involved in actin remodeling, has been shown to cause deafness in mice. While both inner and outer hair cells from Eps8 knockout (KO) mice showed abnormally short stereocilia, inner hair cells (IHCs) also failed to acquire mature-type ion channels. Despite the fact that Eps8 is also expressed in vestibular hair cells, Eps8 KO mice show no vestibular deficits. In the present study we have investigated the properties of vestibular Type I and Type II hair cells in Eps8-KO mice and compared them to those of cochlear IHCs. In the absence of Eps8, vestibular hair cells show normally long kinocilia, significantly shorter stereocilia and a normal pattern of basolateral voltage-dependent ion channels. We have also found that while vestibular hair cells from Eps8 KO mice show normal voltage responses to injected sinusoidal currents, which were used to mimic the mechanoelectrical transducer current, IHCs lose their ability to synchronize their responses to the stimulus. We conclude that the absence of Eps8 produces a weaker phenotype in vestibular hair cells compared to cochlear IHCs, since it affects the hair bundle morphology but not the basolateral membrane currents. This difference is likely to explain the absence of obvious vestibular dysfunction in Eps8 KO mice.

Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia.

SummaryWHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.

Embryonic development of the saccular sensory epithelium in relation to otolith growth in the inner ear of the silver carp, Hypophthalmichthys molitrix (Valenciennes, 1844) (Teleostei: Cyprinidae): light and electron microscopic study

The development of the saccular sensory epithelium in the inner ear of the silver carp, Hypophthalmichthys molitrix, was studied from an early stage (2 days after fertilization), when the otic vesicle or otocyst first formed, to the post-larval stage (7 days after hatching), when the development of the inner ear approximates that of the juvenile stage. Light microscopy revealed: (1) the otic vesicle, the primordia of the saccular otolith and the stato-acoustic ganglion were observed by 2 days after fertilization; (2) the saccular macula overlain by a small round otolith is established at 3 days after fertilization, while the utricular macula with its otolith is first observed at 4 days after fertilization; (3) the saccular macula began to differentiate at 3 days after hatching and became well differentiated at 7 days after hatching. At 7 days after hatching, transmission electron microscopy (TEM) revealed: (1) the apical surface of each hair cell is covered with a ciliary bundle formed of a long kinocilium and a short bundle of stereocilia. The supporting cells are provided with microridges on their apical surface and seemed to be covered with small and large vesicles, suggesting that they have a secretory function beside the supporting one; (2) secretory materials such as multivesicular bodies, electron dense granules, empty vesicles and cytoplasmic extrusions are observed scattering over the saccular sensory epithelium, which probably contribute to the formation of the otolith and/or otolithic membrane. Using scanning electron microscopy (SEM), two types of hair bundles are distinguished: the first type consisted of numerous short stereocilia and a kinocilium; the second type has a small number of short stereocilia with a very long kinocilium. Most of these results are discussed with special regard to the environmental factors affecting the early development of the inner ear in teleost fishes.

Morphological and Morphometric Studies on the Epididymis of West African Dwarf Buck Goat (Capra hircus).

The present studies were carried out on the epididymis collected,in summer season, from twenty-four matured West African Dwarf male goats at the Ipata abattoir, Ilorin, Kwara state, Nigeria. Macroscopically, the results revealed three major regions of the epididymis namely the head, body and tail. The head appeared irregularly oval and flat; the body was long, slender and cylindrical while the tail appeared short and U-shaped. The microscopic examination revealed predominant principal cells, the basal cells, in all regions. The head was characterized by tall columnar epithelium with apical cells;thin long narrow cells and stereocilia, extending into lumen, which was scarcely filled with spermatozoa. The body was characterized by low columnar epithelium with short narrow cells and stereocilia. Its lumen was filled with more spermatozoa than the head region. The tail was formed by cuboidal epithelium with no stereocilia but more densely filled with spermatozoa than the head and body regions. Gross morpho- metric results revealed no significant differences in the mean weight, length of the head, body, tail and total length of the right and left epididymis (p < 0.05). Histo- morphometric result showed that there was a significant reduction in epididymal epithelia height of the tail compared with the head of the right and left epididymis (p < 0.05).

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

High-Speed Hopping Probe Scanning Ion Conductance Microscopy

Vertically protruding stereocilia on the apical surface of the inner ear hair cells represent the ultimate challenge for scanning probe imaging. We have previously imaged these structures, which are largely unresolvable by optical microscopy, using hopping probe scanning ion conductance microscopy (HPSICM) in glutaraldehyde-fixed mammalian auditory hair cells (Novak et al. Nat Methods, 2009). However, it required a considerable amount of time (∼44 min per bundle of ∼8x8 μm). To study live cells, we needed to significantly increase the speed of imaging.For Z-movement we have now used a faster piezo assembly with a resonant frequency of ∼18 kHz, similar to the ones we used for hair bundle deflection. Despite having a less sensitive strain gauge sensor, the vertical resolution of the system remained the same (∼5nm). After adjusting the proportional-integral-derivative controller of the Z-scanner (∼50μs delay) and increasing the speed of approach, we were able to obtain high-resolution images of live hair cell bundles at a frame rate of 12 min/bundle or less.We tested the performance of the improved HPSICM system in live rat inner hair cells (IHC) and showed, for the first time in live cells, the presence of characteristic stereocilia features at an X-Y resolution of ∼11nm. We also imaged IHC bundles from the Shaker2 and Whirler mice due to their short stereocilia with abundant stereocilia links (typically ∼5nm in diameter and ∼100-300nm in length). We confirmed the reproducibility of links in continuous time-lapse scanning and also their absence after chemical disruption with BAPTA-buffered Ca2+-free medium.Our results demonstrate that the improved HPSICM technique successfully visualizes the extremely convoluted surface of stereocilia in live auditory hair cells at a high resolution and a faster speed.Supported by NIDCD/NIH (R01DC008861).

Light microscope observations on the epididymis of paca (Agouti paca).

The features of paca epididymis, based on its appearance in light microscope, is described in this paper. The cellular population of the epithelial lining comprises principal cells, basal cells, apical cells, narrows cells, and hallo cells. The epididymis is divided in five distinct and continuous regions, Zone I, or initial segment, and zone II, are both localized into the head. Zone III comprises the distal head and all the body. Zones IV and V are restricted to the tail, in the proximal and distal cauda epididymis respectively. Each zone can be readily distinguished on the basis of morphological characteristics. The height of epididymal epithelium is greater in zone I. There is a progressive increase in the diameter of the tubular lumen through the different areas, with the maximum in the zone V. The presence of a high epithelium, and the virtual absence of sperm in zone I suggest fast transit of spermatozoa in this region. Zone V comprises the distal tail, has smaller epithelial lining, greater luminal diameter, shorter stereocilia than the other zones, and contains spermatozoa packed inside the lumen, that characterizes this zone as a place of sperm storage. The findings are compared with other reports in rodents and other domestic animals, to contribute to the understanding of epididymal morphophysiology.

Imaging Stereocilia Links in Live Auditory Hair Cells

We previously imaged stereocilia links in glutaraldehyde-fixed mammalian auditory hair cells using hopping probe scanning ion conductance microcopy (HPSICM, Novak et al. Nat Methods, 2009). Due to the complex three-dimensional structure of the stereocilia bundle, achieving high-resolution images required a considerable amount of time (∼44 min/bundle). To study the dynamics of stereocilia link regeneration in real time, we needed a significantly faster yet reliable way to continuously scan the stereocilia bundles in live hair cells.To improve the HPSICM imaging speed, we re-designed the scan head by incorporating a faster piezo assembly with a resonant frequency of ∼18 kHz for Z-movement. Despite having a less sensitive strain gauge sensor (compared to the previously used capacitive sensor), the vertical resolution of the system remained the same (∼5nm). Significantly smaller inertia allowed mounting the scan head on a rotational platform and scanning the sample at any angle, a pre-requisite for the successful imaging of tip links. Moreover, the overall image resolution was slightly decreased and we can now image hair cell bundles significantly faster (∼11 min/bundle).The performance of the improved system was tested using cultured organ of Corti explants from the Shaker 2 and Whirler mice due to their short stereocilia with abundant stereocilia links (typically ∼5nm in diameter and ∼100-300nm in length). To test the ability of HPSICM to detect these miniature structures at high imaging speed we performed continuous time-lapse scanning and looked for reproducibility of the links in consecutive images. Next, to rule out the possibility that the observed links were simply scanning artifacts, we disrupted the links by treating the explants with BAPTA-buffered Ca2+-free medium. Our results demonstrate that the improved HPSICM technique successfully visualizes stereocilia links in live auditory hair cells.Supported by NIDCD/NIH (ARRA supplement to R01DC008861)

Coupling of the mechanotransduction machinery and stereocilia F-actin polymerization in the cochlear hair bundles

Mechanoelectrical transduction (MET), the conversion of mechanical stimuli into electrical signals operated by the sensory cells of the inner ear, enables hearing and balance perception. Crucial to this process are the tip-links, oblique fibrous filaments that interconnect the actin-filled stereocilia of different rows within the hair bundle, and mechanically gate MET channels. In a recent study, we observed a complete regression of stereocilia from the short and medium but not the tall row upon the disappearance of the tip-links caused by the loss of one of their components, cadherin-23, or of one of their anchoring proteins, sans, in the auditory organs of engineered mutant mice. This indicates the existence of a coupling between the MET and F-actin polymerization machineries at the tips of the short and medium stereocilia rows in cochlear hair bundles. Here, we first present our findings in the mutant mice, and then discuss the possible effects of the tip-link tension on stereocilia F-actin polymerization, acting either directly or through Ca(2+)-dependent mechanisms that involve the gating of MET channels.

Regulation of Stereocilia Length by Myosin XVa and Whirlin Depends on the Actin-Regulatory Protein Eps8

Myosin XVa (MyoXVa) and its cargo whirlin are implicated in deafness and vestibular dysfunction and have been shown to localize at stereocilia tips and to be essential for the elongation of these actin protrusions [1-4]. Given that whirlin has no known actin-regulatory activity, it remains unclear how these proteins work together to influence stereocilia length. Here we show that the actin-regulatory protein Eps8 [5] interacts with MyoXVa and that mice lacking Eps8 show short stereocilia compared to MyoXVa- and whirlin-deficient mice. We show that Eps8 fails to accumulate at the tips of stereocilia in the absence of MyoXVa, that overexpression of MyoXVa results in both elongation of stereocilia and increased accumulation of Eps8 at stereocilia tips, and that the exogenous expression of MyoXVa in MyoXVa-deficient hair cells rescues Eps8 tip localization. We find that Eps8 also interacts with whirlin and that the expression of both Eps8 and MyoXVa at stereocilia tips is reduced in whirlin-deficient mice. We conclude that MyoXVa, whirlin, and Eps8 are integral components of the stereocilia tip complex, where Eps8 is a central actin-regulatory element for elongation of the stereocilia actin core.

MyosinVIIa Interacts with Twinfilin-2 at the Tips of Mechanosensory Stereocilia in the Inner Ear

In vertebrates hearing is dependent upon the microvilli-like mechanosensory stereocilia and their length gradation. The staircase-like organization of the stereocilia bundle is dynamically maintained by variable actin turnover rates. Two unconventional myosins were previously implicated in stereocilia length regulation but the mechanisms of their action remain unknown. MyosinXVa is expressed in stereocilia tips at levels proportional to stereocilia length and its absence produces staircase-like bundles of very short stereocilia. MyosinVIIa localizes to the tips of the shorter stereocilia within bundles, and when absent, the stereocilia are abnormally long. We show here that myosinVIIa interacts with twinfilin-2, an actin binding protein, which inhibits actin polymerization at the barbed end of the filament, and that twinfilin localization in stereocilia overlaps with myosinVIIa. Exogenous expression of myosinVIIa in fibroblasts results in a reduced number of filopodia and promotes accumulation of twinfilin-2 at the filopodia tips. We hypothesize that the newly described interaction between myosinVIIa and twinfilin-2 is responsible for the establishment and maintenance of slower rates of actin turnover in shorter stereocilia, and that interplay between complexes of myosinVIIa/twinfilin-2 and myosinXVa/whirlin is responsible for stereocilia length gradation within the bundle staircase.

Ultrastructure and distribution of superficial neuromasts of blind cavefish, Phreatichthys andruzzii, juveniles

Transmission and scanning electron microscopy (TEM, SEM) were used to study the ultrastructure of superficial neuromasts in 15 six-month old blind cavefish juveniles, Phreatichthys andruzzii (Cyprinidae). In five specimens examined with SEM, the number of superficial neuromasts over the fish body (480-538) was recorded. They were localized mainly on the head (362-410), including the dorsal surface, the mentomandibular region, and laterally from the mouth to the posterior edge of the operculum. Neuromasts were also present laterally on the trunk and near the caudal fin (116-140). A significantly higher number of neuromasts were present on the head compared to the trunk (t-test, P < 0.05). Superficial neuromasts of the head and those along the trunk were similar in ultrastructure. Each neuromast comprised sensory hair cells surrounded by nonsensory support cells (mantle cells and supporting basal cells) with the whole covered by a cupula. Each hair cell was pear-shaped, 15-21 microm high and 4-6 microm in diameter, with a single long kinocilium and several short stereocilia. Most support cells were elongated, with nuclei occupying a large portion of the cytoplasm. In the margin of the neuromast, mantle cells were particularly narrow. Both types of support cells had well-developed Golgi apparatus and rough endoplasmic reticulum. The number of hair cells and nonsensory support cells of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (t-test, P > 0.05).

Fast Adaptation and Ca2+Sensitivity of the Mechanotransducer Require Myosin-XVa in Inner But Not Outer Cochlear Hair Cells

In inner ear hair cells, activation of mechanotransduction channels is followed by extremely rapid deactivation that depends on the influx of Ca(2+) through these channels. Although the molecular mechanisms of this "fast" adaptation are largely unknown, the predominant models assume Ca(2+) sensitivity as an intrinsic property of yet unidentified mechanotransduction channels. Here, we examined mechanotransduction in the hair cells of young postnatal shaker 2 mice (Myo15(sh2/sh2)). These mice have no functional myosin-XVa, which is critical for normal growth of mechanosensory stereocilia of hair cells. Although stereocilia of both inner and outer hair cells of Myo15(sh2/sh2) mice lack myosin-XVa and are abnormally short, these cells have dramatically different hair bundle morphology. Myo15(sh2/sh2) outer hair cells retain a staircase arrangement of the abnormally short stereocilia and prominent tip links. Myo15(sh2/sh2) inner hair cells do not have obliquely oriented tip links, and their mechanosensitivity is mediated exclusively by "top-to-top" links between equally short stereocilia. In both inner and outer hair cells of Myo15(sh2/sh2) mice, we found mechanotransduction responses with a normal "wild-type" amplitude and speed of activation. Surprisingly, only outer hair cells exhibit fast adaptation and sensitivity to extracellular Ca(2+). In Myo15(sh2/sh2) inner hair cells, fast adaptation is disrupted and the transduction current is insensitive to extracellular Ca(2+). We conclude that the Ca(2+) sensitivity of the mechanotransduction channels and the fast adaptation require a structural environment that is dependent on myosin-XVa and is disrupted in Myo15(sh2/sh2) inner hair cells, but not in Myo15(sh2/sh2) outer hair cells.

The Lateral Line System in Larvae of the Blind Cyprinid Cavefish, Phreatichthys andruzzii

Scanning and transmission electron microscopic observations were made on the anterior and posterior lateral line system of larvae of the blind cavefish, Phreatichthys andruzzii. The investigations were conducted on 36 specimens belonging to three different ages of juvenile: 7, 14, and 28 days posthatching. The basic sensory unit of the mechanoreceptive system is a superficial "neuromast," which is composed of sensory hair cells (HCs) embedded in nonsensory support cells (SCs) frequently covered with a cupula. Head neuromasts had the longest cupulae. These organs presented the same structure and ultrastructure in the head and along the body of all specimens of P. andruzzii of different ages. Hair cells of neuromasts of P. andruzzii larvae were pear-shaped cells approximately 10-20 microm high and 3-5 microm in diameter. The nucleus of each hair cell was large and basally placed, the cytoplasm contained numerous mitochondria and each HC had a single long kinocilium and several short stereocilia. The majority of SCs were elongated in shape and their nuclei occupied a significant portion of the cytoplasm. The support cells at the margin of the neuromast were particularly thin. Both types of support cells possessed well-developed rough endoplasmic reticulum and Golgi apparatus. The size and number of neuromasts and their component cells increased with the age of larvae (ANOVA, P < 0.05). The dimensions of the neuromasts of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (ANOVA, P > 0.05).