In the lateral line system, water motion is detected by neuromast organs, fundamental units that are arrayed on a fish’s surface. Each neuromast contains hair cells, specialized mechanoreceptors that convert mechanical stimuli, in the form of water movement, into electrical signals. The orientation of hair cells’ mechanosensitive structures ensures that the opening of mechanically-gated channels is maximal when deflected in a single direction. In each neuromast organ, hair cells have two opposing orientations, enabling bi-directional detection of water movement. Interestingly, Tmc2b and Tmc2a proteins, which constitute the mechanotransduction channels in neuromasts, distribute asymmetrically so that Tmc2a is expressed in hair cells of only one orientation. Here, using both in vivo recording of extracellular potentials and calcium imaging of neuromasts, we demonstrate that hair cells of one orientation have larger mechanosensitive responses. The associated afferent neuron processes that innervate neuromast hair cells faithfully preserve this functional difference. Moreover, Emx2, a transcription factor required for the formation of hair cells with opposing orientations, is necessary to establish this functional asymmetry within neuromasts. Remarkably, loss of Tmc2a does not impact hair cell orientation but abolishes the functional asymmetry as measured by recording extracellular potentials and calcium imaging. Overall, our work indicates that oppositely oriented hair cells within a neuromast employ different proteins to alter mechanotransduction to sense the direction of water motion.

Calcium imaging

Acquisition:

GCaMP6s-based calcium imaging of zebrafish hair bundles and of the afferent processes (day 4-6). A fluid jet was used to mechanically stimulate the apical bundles of hair cells of the A-P (L1-L4) neuromasts. To stimulate the two orientations of hair cells (A-to-P sensitive and P-to-A sensitive) a 500-ms ‘push’ was delivered. Larvae were rotated 180° to deliver a comparable ‘push’ stimulus to both the A-to-P sensitive and P-to-A sensitive cells. To image calcium-dependent mechanosensation in apical hair bundles or calcium-dependent activity in the afferent process, a Bruker swept-field confocal system was used. The system was equipped with a Rolera EM-C2 CCD camera (QImaging) and a Nikon CFI Fluor 60✕ 1.0 NA water immersion objective. Images were acquired in 5 planes along the Z-axis at 0.5 mm intervals (hair bundles) or 1 mm intervals (afferent processes), at a 50 Hz frame rate yielding a 10 Hz volume rate.

Processing:

The 5 plane Z-stacks were projected into one plane for image processing and quantification. For GCaMP6s measurements, a circular ROI with a ~1.5 mm (hair bundles) or 3 mm (afferent processes) diameter was placed on the center of each individual bundle or afferent hot spot. The mean intensity (∆F/F₀) within each ROI was quantified where F₀ represents the GCaMP6s intensity prior to stimulation. The GCaMP6s responses for each neuromast were averaged to quantify the magnitude of the A-to-P sensitive and P-to-A sensitive responses.

Immunohistochemistry

Acquisition:

Immunohistochemistry to label myosin7a, Emx2 and actin or synapses was performed on whole zebrafish larvae. The following primary antibodies were used: rabbit anti-myosin7a (Proteus 25-6790; 1:500); mouse anti-Emx2 (Trans Genic KO609; 1:250); mouse anti-pan-MAGUK (IgG1) (Millipore MABN7; 1:500); and mouse anti-Ribeye b (IgG2a) (Sheets et al 2011; 1:10,000). The following secondary antibodies were used at 1:1000: (#A11010, #A21236, #A-11008, #A-21133, #A-21240, ThermoFisher Scientific) along with Alexa 488 Phalloidin (#A12379, ThermoFisher Scientific). Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using an 63✕ 1.4 NA oil objective lens.

Synapse quantification:

Myosin7a and phalloidin label were used in combination to link hair bundles to their respective cell base and synapses. To qualify as a ribbon or postsynapse, the following minimum size filters were applied: Ribeye b: 0.025 μm², pan-Maguk: 0.04 μm². A complete synapse was comprised of both Ribeye b and MAGUK puncta. In each neuromast, synapses in four A-to-P and four P-to-A cells were examined for quantification.

• FIJI (to open calcium imaging and immunostain data)
• Prism 8 (to open spreadsheets)