DOI: 10.5061/dryad.jm63xsjg2
title: Asymmetric mechanotransduction by hair cells of the zebrafish lateral line (Part 1/2)

description: 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.
description: 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/F<sub>0</sub>) within each ROI was quantified where
 F<sub>0</sub> 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 μm<sup>2</sup>, pan-Maguk: 0.04
 μm<sup>2</sup>. 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.
description: FIJI (to open calcium imaging and immunostain
 data) Prism 8 (to open spreadsheets)