Organization of the superficial neuromast system in goldfish, Carassius auratus

Distribution, morphology, and orientation of superficial neuromasts and polarization of the hair cells within superficial neuromasts of the goldfish (Carassius auratus) were examined using fluorescence labeling and scanning electron microscopy. On each body side, goldfish have 1,800-2,000 superficial neuromasts distributed across the head, trunk and tail fin. Each superficial neuromast had about 14-32 hair cells that were arranged in the sensory epithelium with the axis of best sensitivity aligned perpendicular to the long axis of the neuromast. Hair cell polarization was rostro-caudal in most superficial neuromasts on trunk scales (with the exception of those on the lateral line scales), or on the tail fin. On lateral line scales, the most frequent hair cell polarization was dorso-ventral in 45% and rostro-caudal in 20% of the superficial neuromasts. On individual trunk scales, superficial neuromasts were organized in rows which in most scales showed similar orientations with angle deviations smaller than 45 degrees . In about 16% of all trunk scales, groups of superficial neuromasts in the dorsal and ventral half of the scale were oriented orthogonal to each other. On the head, most superficial neuromasts were arranged in rows or groups of similar orientation with angle deviations smaller than 45 degrees . Neighboring groups of superficial neuromasts could differ with respect to their orientation. The most frequent hair cell polarization was dorso-ventral in front of the eyes and on the ventral mandible and rostro-caudal below the eye and on the operculum.     

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.     

Paired development of hair cells in neuromasts of the teleost lateral line.

Lateral line neuromasts of the bullseye Parapriacanthus ransonetti and the cardinal fish Apogon cyanosoma were examined by scanning electron microscopy. Neuromasts showed large numbers of degenerating hair cells and immature hair cells, suggesting a high degree of hair cell turnover. New hair cells were mainly produced in pairs (fewer than 5% appear singly), the two cells of a pair having opposite but parallel orientations of their mechanosensitive axes. It is suggested that each pair results, directly or indirectly, from a single mitosis. The results further suggest that the axis of mitosis is one of the factors which determine the direction of the hair cell axis of mechanosensitivity.     

Development of Free Neuromasts with Special Reference to Sensory Polarity in Larvae of the Willow Shiner, Gnathopogon elongatus caerulescens (Teleostei, Cyprinidae)

To find how larval fish sense mechanical stimuli via their free neuromasts, we examined morphological changes in free neuromasts in the larval willow shiner, Gnathopogon elongatus caerulescens. Free neuromasts were found on the body surface of newly hatched larvae and their number increased on both the head and trunk with larval growth. The apical surface of free neuromasts changed in outline from a circle to a lozenge shape as the number of sensory cells increased in the prelarval stage, and then the cupulae of the free neuromasts changed from a stick-like to a blade-like shape. Seven-day-old larvae were at the postlarval stage and had many free neuromasts that were nearly mature. All free neuromasts contained sensory cells of opposing polarity. The orientation of the maximum sensitivity of free neuromasts, decided from the polarity of the sensory cells, coincided with the minor axis of the lozenge-shaped outline of the apical surface of the free neuromasts, and was in the same axis as the direction in which the blade-like cupulae bent. The change to a blade-like shape would cause a stimulus parallel to the minor axis to be perceived as being stronger than the same stimulus from other directions. The polarity of trunk neuromasts was usually oriented along the antero-posterior axis of the fish body, but a few had dorso-ventral orientation. On the head, free neuromasts were oriented on lines tangential to concentric circles around the eye.     

VISION AND SENSORY PHYSIOLOGY The lateral line systems of three deep-sea fish

The distribution and ultrastructure of the lateral line systems in three taxonomically dispersed deep-sea fish are described: Poromitra capito, Melanonus zugmayeri and Phrynichthys wedli. They are meso- to bathpelagic and are thought to feed on small crustaceans and fish. All possess highly developed lateral line systems, a feature associated with life in the deep sea. Poromitra capito and M. zugmayeri exhibit widened head canals which are connected to the outside by large pores and which contain around 60 large neuromasts. Each neuromast consists of a cupula, shield-shaped mantle and a sensory plate containing hundreds to thousands of hair cells. Direction of sensitivity is in the long axis of the canal (perpendicular to the long axis of the mantle). Depending on their position on the sensory plate, the hair cells have different morphologies. They fall into three basic classes which, from comparison with past work, may be tuned to different frequencies. Alternatively, the various hair cell morphologies could be interpreted as being members of a developmental or growth sequence. Phrynichthys wedli has no canal organs, these being replaced secondarily by many superficial neuromasts placed on prominent papillae in rows which cover much of the 'head' and body. Direction of sensitivity is along the axis of the neuromast row. An extreme proliferation of superficial neuromasts are also found on the heads of P. capito and M. zugmayeri and these are of a type not described before. They consist of stitches, raised on papillae in M. zugmayeri and several mm long in P. capito , in which continuous lines of hair cells, two to three cells wide, are embedded. Direction of sensitivity is perpendicular to the long axis of the stitch. Based on the structure and direction of sensitivity, possible functional implications of all the neuromast types described are compared and discussed.     

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.     

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.     

Development of lateral line organs in leptocephali of the freshwater eel Anguilla japonica (Teleostei,Anguilliformes)

A study of the ontogeny of the lateral line system in leptocephali of the Japanese eel Anguilla japonica reveals the existence of three morphologically different types of lateral line organs. Type I is a novel sensory organ with hair cells bearing a single kinocilium, lacking stereocilia, distributed mainly on the head of larvae, and morphologically different from typical superficial neuromasts of the lateral line system. Its developmental sequence suggests that it may be a presumptive canal neuromast. Type II is an ordinary superficial neuromast, common in other teleost larvae, which includes presumptive canal neuromasts that first appear on the trunk and accessory superficial neuromasts that later appear on the head and trunk. Type III is a very unusual neuromast located just behind the orbit, close to the otic vesicle, with radially oriented hair cells, suggesting that these serve as multiple axes of sensitivity for mechanical stimuli. The behavior of larval eels suggests that the radially oriented neuromasts may act as the sole mechanosensory organ until the ordinary superficial neuromasts develop. The finding that larval eels possess a well-developed mechanosensory system suggests the possibility that they are also capable of perceiving weak environmental mechanical stimuli, like other teleost larvae.     

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.     

Ultrastructure of the lateral-line sense organs of the ratfish,Chimaera monstrosa

Summary The ultrastructure of the lateral-line neuromasts in the ratfish, Chimaera monstrosa is described. The neuromasts rest at the bottom of open grooves and consist of sensory, supporting, basal and mantle cells. Each sensory cell is equipped with sensory hairs consisting of a single kinocilium and several stereocilia. There are several types of sensory hair arrangement, and cells with a particular arrangement form patches within the neuromast. There are two types of afferent synapse. The most common afferent synapse has a presynaptic body and is typically associated with an extensive system of anastomosing tubules on the presynaptic side. When the tubules are absent, vesicles surround the presynaptic body. These synapses are often associated into synaptic fields, containing up to 35 synaptic sites. The second type of afferent synapse does not have a presynaptic body and is not associated with the tubular system. The afferent synapses of the second type do not form synaptic fields and are uncommon. The efferent synapses are either associated with a postsynaptic sac or more commonly with a strongly osmiophilic postsynaptic membrane. The accessory cells are similar to those in the acoustico-lateralis organs of other aquatic vertebrates. A possibility of movement of the presynaptic bodies and of involvement of the tubular system in the turnover of the transmitter is discussed. A comparison of the hair tuft types in the neuromasts of Ch. monstrosa with those in the labyrinth of the goldfish and of the frog is attempted.     

Morphogenetic tissue movement and the establishment of body plan during development from blastocyst to gastrula in the mouse

In many animal species, the early development of the embryo follows a stereotypic pattern of cell cleavage, lineage allocation and generation of tissue asymmetry leading to delineation of the body plan with three primary embryonic axes. The mammalian embryo has been regarded as an exception and primary body axes of the mouse embryo were thought to develop after implantation. However, recent findings have challenged this view. Asymmetry in the fertilised oocyte, as defined by the position of the second polar body and the sperm entry point, can be correlated with the orientation of the animal-vegetal and the embryonic-abembryonic axes in the preimplantation blastocyst. Studies of the pattern of morphogenetic movement of cells and genetic activity in the peri-implantation embryo suggest that the animal-vegetal axis of the blastocyst might presage the orientation of the anterior-posterior axis of the gastrula. This suggests that the asymmetry of the zygote that is established at fertilisation and early cleavage has a lasting impact on the delineation of body axes during embryogenesis.     

Ultrastructure of free neuromasts of Bathygobius fuscus (gobiidae) and canal neuromasts of Apogon cyanosoma (apogonidae)

Light and electron microscopic observations were made on the lateral line organs of the free neuromasts of the goby Bathygobius fuscus and the canal neuromasts of the cardinal fish Apogon cyanosoma. As in other lateral line systems, each neuromast consists of hair cells, supporting cells and mantle supporting cells, the whole being covered by a cupula. In B. fuscus the free neuromasts are mounted on papillae and have hair cells with stereocilia up to 2.5 μm long and a single kinocilium at least 25 μm long. Each neuromast is covered by a vane-like cupula that can be divided into two regions. The central region over the sensory area contains columns of myelin-like figures. These figures are absent from the outer region covering the mantle. The canal neuromasts of A. cyanosoma are diamond-shaped with up to 1,500 hair cells. The cupula is unusual in having a channel that lies over the sensory region. The hair cells have up to 45 stereocilia, the tallest reaching 2.5 μm, and a kinocilium at least 5 μm long. Tip links are shown for the first time between rows of stereocilia of the hair cells of lateral line neuromasts. The presence of tip links has now been demonstrated for all acousticolateral hair cell systems.     

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.     

Coding of acoustic particle motion by utricular fibers in the sleeper goby, <Emphasis Type="Italic">Dormitator latifrons</Emphasis>

It is unknown whether the fish utricle contributes to directional hearing. Here, we report response properties of single utricular fibers in a teleost fish (Dormitator latifrons) to linear accelerations at various stimulus frequencies and axes. Characteristic frequencies ranged from 50–400 Hz (median=80 Hz), and best frequencies shifted from 50 to 250 Hz with stimulus level. Best sensitivity of utricular fibers was distributed from –70 to –40 dB re: 1 g (mean=–52 dB), which is about 30 dB less sensitive than saccular fibers. Q_50% fell between 0.16 and 11.50 (mean=2.04) at 15 dB above threshold. We observed temporal response patterns of entrained phase-locking, double phase-locking, phase-locked bursting, and non-phase-locked bursting. Most utricular fibers were directionally selective with various directional response profiles, and directional selectivity was stimulus-level dependent. Horizontal best-response axes were distributed in a 152° range while mid-sagittal best-response axes were clustered around the fish longitudinal axis, which is consistent with the horizontal orientation of the utricle and morphological polarizations of utricular hair cells. Therefore, results of this study indicate that the utricle in this vertebrate plays an auditory role in azimuth and that utricular fibers extend the response dynamic range of this species in directional hearing.     

Pit organs in axolotls: a second class of lateral line neuromasts

The lateral line system of axolotls (Ambystoma mexicanum) consists of mechanoreceptive neuromasts and electroreceptive ampullary organs. All neuromasts in salamanders are located superficially and are organized into lines that are homologous to canal neuromasts in fishes. Ampullary organs are confined to the head and generally are located adjacent to the lines of superficial neuromasts. Axolotls, however, also possess a third class of receptors; these form restricted patches on the head and are possibly homologous to the superficial pit organs in fishes. In order to test this hypothesis the morphology of the suspected pit organs was examined with scanning electron microscopy, and a number of their physiological properties were determined. Pit organs are approximately half the size of neuromasts and have fewer hair cells, although these hair cells do possess kinocilia and stereocilia like those of neuromasts. Pit organs also possess cupulae and exhibit a pattern of innervation identical to that of neuromasts. Pit organs and neuromasts also exhibit similar rates of spontaneous activity, are excited by weak water currents but not weak electric stimuli, and are not inhibited by magnesium ions. Pit organs appear to have slightly lower rates of spontaneous discharge than neuromasts, however, and have slightly lower displacement thresholds to low frequency wave stimuli. These data support the contention that the pit organs of axolotls constitute a second class of neuromasts homologous to the pit organs of fishes.     

Morphogenesis of free neuromasts in the larvae of brown-marbled grouper Epinephelus fuscoguttatus

Newly hatched larvae had one pair of free neuromasts behind the eyes. As the larvae grew, free neuromasts increased in number. The apical surface of sensory epithelium widened and subsequently elongated. The number of sensory hair cells increased and the directions of maximum sensitivity became both anteroposterior and dorsoventral on the trunk. Before notochord flexion, only the anteroposterior type was observed. After notochord flexion, two types of neuromasts were observed on the trunk. On the head, the orientation of free neuromasts formed a tangential line to concentric circles around the eyes and nostrils. Free neuromasts on the head could therefore receive stimuli from various angles from predators or zooplanktons. This suggests that these free neuromasts play a role in compensating for a dead angle of vision, and an important role in detecting zooplankton under scotopic vision. Canal organs were observed on the head and operculum in 40-d-old animals.     

Directional encoding by fish auditory systems

This paper reviews and discusses several investigations of the peripheral neural code for the directional axis of acoustical particle motion in the saccule of two fishes: goldfish (Carassius auratus) and toadfish (Opsanus tau). Most saccular afferents are directional in the manner of hair cells, having a cosine-shaped directional response pattern. The saccular sensory epithelia are orientated almost vertically in a parasagittal plane. In the horizontal plane, these epithelia are orientated obliquely with respect to the midline. Hair-cell stereocilia project perpendicularly. Thus, directional response patterns of saccular afferents tend to be orientated in azimuth parallel to the orientation of the epithelia in the head. The oblique angle of the toadfish saccule is greater than that of the goldfish, and the range of best directions in the horizontal plane for each species reflects those differing orientations. The azimuth of acoustical particle motion could be computed by comparing the relative activation of the two saccules, as is the case for the ears of most terrestrial vertebrates. The spatial patterns of saccular hair-cell orientation of most fishes thus appear to have little function in azimuthal source location, but for toadfish are probably most important for determining the elevation of monopole sources.     

Neuromast formation in the prehatching embryos of the Japanese flounder (Paralichthys olivaceus)

The present paper clarifies the initial development of the lateral line organs in the embryonic Japanese flounder, Paralichthys olivaceus. The first appearances of lateral line primordia, and the proliferation, distribution and morphological development of the free neuromasts, including nerve ending formation: establishment of hair cell innervations via the formation of synapses, were examined by light microscopy, scanning and transmission electron microscopy. The first pair of neuromast primordia appeared in the otic region ≈ 30 h prior to hatching and subsequently differentiated into free neuromasts, otic neuromasts, after ≈ 8 h. At hatching, a pair of free neuromasts and three pairs of neuromast primordia were present on the head, and three pairs of neuromast primordia were present on the trunk. The hair cell polarity of the otic neuromast until just prior to hatching was radial, but not bi‐directional. The typical afferent and efferent nerve endings in the otic neuromasts had formed by the time of hatching, suggesting that the otic neuromasts are functional prior to hatching. The three neuromast primordia located on each side of the trunk were derived from a long, narrow ectodermal cell cluster and erupted through the epidermis after hatching.     

The lateral line receptor array of cyprinids from different habitats

The lateral line system of teleost fishes consists of an array of superficial and canal neuromasts (CN). Number and distribution of neuromasts and the morphology of the lateral line canals vary across species. We investigated the morphology of the lateral line system in four diurnal European cyprinids, the limnophilic bitterling (Rhodeus sericeus), the indifferent gudgeon (Gobio gobio), and ide (Leuciscus idus), and the rheophilic minnow (Phoxinus phoxinus). All fish had lateral line canals on head and trunk. The total number of both, CN and superficial neuromasts (SN), was comparable in minnow and ide but was greater than in gudgeon and bitterling. The ratio of SNs to CNs for the head was comparable in minnow and bitterling but was greater in gudgeon and ide. The SN‐to‐CN ratio for the trunk was greatest in bitterling. Polarization of hair cells in CNs was in the direction of the canal. Polarization of hair cells in SNs depended on body area. In cephalic SNs, hair cell polarization was dorso‐ventral or rostro‐caudal. In trunk SNs, it was rostro‐caudal on lateral line scales and dorso‐ventral on other trunk scales. On the caudal fin, hair cell polarization was rostro‐caudal. The data show that, in the four species studied here, number, distribution, and orientation of CNs and SNs cannot be unequivocally related to habitat. J. Morphol. 275:357–370, 2014. © 2013 Wiley Periodicals, Inc.     

Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms

Auditory hair cells represent one of the most prominent examples of epithelial planar polarity. In the auditory sensory epithelium, planar polarity of individual hair cells is defined by their V-shaped hair bundle, the mechanotransduction organelle located on the apical surface. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue planar polarity is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the hair cell-intrinsic polarity machinery that establishes the V-shape of the hair bundle is poorly understood. Here, we show that the microtubule motor subunit Kif3a regulates hair cell polarization through both ciliary and non-ciliary mechanisms. Disruption of Kif3a in the inner ear led to absence of the kinocilium, a shortened cochlear duct and flattened hair bundle morphology. Moreover, basal bodies are mispositioned along both the apicobasal and planar polarity axes of mutant hair cells, and hair bundle orientation was uncoupled from the basal body position. We show that a non-ciliary function of Kif3a regulates localized cortical activity of p21-activated kinases (PAK), which in turn controls basal body positioning in hair cells. Our results demonstrate that Kif3a-PAK signaling coordinates planar polarization of the hair bundle and the basal body in hair cells, and establish Kif3a as a key component of the hair cell-intrinsic polarity machinery, which acts in concert with the tissue polarity pathway.