Organization of neuromasts and their innervation.

(A) A schematic diagram depicts the posterior lateral line of the larval zebrafish. While migrating from the ear to the tip of the tail, the first lateral-line primordium deposits about seven neuromasts (yellow, L1 through L6 and 7). The hair cells of these neuromasts are uniformly polarized along the anteroposterior axis; a second primordium subsequently deposits a few neuromasts (not shown) with a dorsoventral polarity. Individual neurons of the posterior lateral-line ganglion, one of which is portrayed, are innervated by hair cells in one to five successive neuromasts and convey information into the brainstem. (B) A schematic diagram of an individual neuromast shows a cluster of hair cells surmounted by a gelatinous cupula into which their hair bundles insert. When water movement (arrow) deflects the cupula, the long kinocilia are bent and their motion is communicated to and transduced by the stereocilia. Half of the hair cells (red) are polarized for sensitivity to caudad movement, the other half (blue) to rostrad motion. The hair cells are separated by supporting cells (lavender) and bounded by mantle cells (green). (C) A high-resolution, deconvolved fluorescence image shows the apical surfaces of hair cells in the plane of section indicated by parallel dashed lines in panel (B). The cuticular plate at the apex of each hair-cell soma is marked by actin-GFP. The dark spot within each cuticular plate represents the base of the kinocilium, which denotes the hair bundle's polarity. (D) A schematic diagram depicts the organization of hair cells in a neuromast of the posterior lateral line. There are roughly equal numbers of hair cells sensitive to caudad water motion (red) and to rostrad movement (blue). As hair cells migrate toward the neuromast's equator (pale cells), they senesce and die. (E) Uncharacterized stem cells located near the apical and basal poles of the neuromast produce transit-amplifying cells, each of which divides into daughter cells that undergo a rotatory rearrangement in about half the instances. After rearrangement has concluded, the nascent hair cells in wild-type larvae always adopt opposite polarities. (F) An individual afferent nerve fiber marked with mCherry forms postsynaptic endings (arrowheads) on three hair cells. (G) An image of the apical surface of the same neuromast shows phalloidin-labeled cuticular plates and hair bundles. Consistent with polarity-specific innervation, the three hair cells contacted by the fiber in panel (F) all display polarization to caudad stimuli. (H) A reconstruction of the innervation of a single neuromast includes afferent terminals, which receive synaptic input from sensory hair cells, and an efferent terminal that innervates hair cells. Details are provided in the caption of Video 2. (I) Steps in serial blockface scanning electron microscopy (SBFSEM) of a lateral-line neuromast. (i). A larva is preserved in aldehyde-based fixative and impregnated with heavy metal to enhance the specimen's contrast and electrical conductivity. (ii) The specimen is embedded in plastic and cut (dotted lines) to isolate the region containing the neuromast of interest (arrowhead). (iii) The trimmed specimen is secured to a mount and placed in the scanning electron microscope. (iv) A diamond knife makes hundreds to thousands of passes across the blockface, progressively scraping away tens of nanometers of the specimen. (v) After each passage of the knife, an electron beam scans the blockface; the emitted secondary electrons are collected to form an image of the sectioned specimen. (J) A typical serial blockface scanning electron micrograph shows several hair cells, whose nuclei (HC) occur in a layer above that encompassing supporting-cell nuclei (SC).

SBFSEM images of neuromast components.

(A) A low-power micrograph shows the general organization of a neuromast. The sensory organ lies between skeletal-muscle fibers, from which it is separated by the epithelial basal lamina, and the aqueous environment, into which the hair bundles protrude. The nuclei of hair cells form a layer above those of supporting cells. (B) The posterior lateral-line nerve (outlined in orange) contains two myelinated axons as well as numerous unmyelinated fibers. Seven afferent terminals, each denoted as ‘A’ and distinctively colored and numbered, arise as single branches of axons in the nerve and enter the neuromast through a pore in the epithelial basal lamina. One efferent axon labeled ‘E’ is also present. (C) The synaptic terminal of an efferent axon onto a hair cell displays a vesicle-filled bouton. Within the hair cell's cytoplasm lies a cistern that sequesters the Ca²⁺ that enters the cell during synaptic activity. (D) A perisynaptic compartment (outlined in orange) contains four afferent terminals, each denoted as ‘A’ and numbered, and a single efferent terminal labeled ‘E.’ The compartment is demarcated by two supporting cells. The hair cell makes an afferent synapse onto the axonal terminal A1. A synaptic ribbon in the hair cell is characterized by moderate electron density and a clear halo surrounded by synaptic vesicles. The ribbon is readily distinguished from the numerous mitochondria in all cell types. In each of the panels, the areas within some of the cellular contours delineated by annotators have been colored to emphasize specific cells or axonal terminals. In order to provide a broad color gamut, the scheme of coloration here is arbitrary and distinct from that in the other figures.