Structure, multiple alignment, and phylogenetic tree of Mucin-5AC proteins. (A) Schematic diagram of Mucin-5AC domains in zebrafish, little skate, spotted gar, medaka, and human. SP, signal peptide; CysD, cysteine rich domain; PTS, central proline, threonine, serine (PTS)-rich sequence. Green bars represent von Willebrand domains. (B) Phylogenetic tree of the first or second CysD domains of Mucin-5ACs. (C) Multiple alignment of the first or second CysD domain of Mucin-5ACs. Asterisks represent conserved cysteines (C). Black boxes indicate conserved serines (S) and threonines (T). (D) Secondary structure element of CysD1 in zebrafish Mucin-5AC. (E) Amino-acid sequence arrangement of CysD1 in zebrafish Mucin-5AC. The blue arrows (β1–β6) represent anti-parallel beta-strands and the yellow cylinders (α1–α2) represent short alpha helices. The black lines (SS1–SS2) mark disulfide bonds. (F) Three-dimensional structure of zebrafish Mucin-5AC (CysD1-CysD4) predicted by Alphafold.

Zebrafish muc5AC is specifically expressed in neuromasts. (A) Representative image of whole-mount in situ hybridization of muc5AC at 3 dpf. Inset is the enlarged image of a neuromast in (A). Dashed line shows the outline of the neuromast. (B) Representative image of whole-mount in situ hybridization of muc5AC at 5 dpf. Scale bar of the inset, 50 μm; Scale bar of (A,B), 500 μm.

Characterization of the expression pattern of Tg(3kmuc5AC:EGFP) and Tg(3kmuc5AC:EGFP). (A) Schematic diagram of the Tol2 construct used to generate the Tg(3kmuc5AC:EGFP) reporter. (B,C) Dorsal view and lateral view of a Tg(3kmuc5AC:EGFP) larva at 5 dpf. Scale bars: 200 μm. (D,E) Head and posterior region of operculum shown by Tg(3kmuc5AC:EGFP) in adult zebrafish. White arrows label canal neuromasts. Scale bars in D and E: 1 mm. (F) Schematic diagram of the Tol2 construct used to generate the Tg(3kmuc5AC:H2A-mCherry) reporter. (G–G”) Immunostaining with anti-mCherry (red) and anti-Sox2 (green). (H–H”) Live imaging of Tg(3kmuc5AC:H2A-mCherry) (red) stained with Yo-Pro-1 (green). (I–I”) Live imaging of Tg(3kmuc5AC:H2A-mCherry) (red) and Tg(ET20:EGFP) (green) double transgenic line. Scale bars: 20 μm.

Knockdown of muc5AC reduces the length of cupulae in zebrafish larvae. (A) Schematic diagram of the target of muc5AC-Mo in muc5AC mRNA. (B,C) Live imaging of the larvae injected with control-Mo (B) and muc5AC-Mo (C) at 5 dpf. The hair cells and cupula are shown by Tg(Brn3c:EGFP) (green) and fluorescent microsphere (red), respectively. Scale bars: 50 μm.

Mutation of muc5AC reduces the length of cupula in zebrafish larvae. (A) Schematic diagrams of muc5AC gene knockout. Top panel shows the scheme of muc5AC gene locus. Blue bars represent coding exon. Black bars represent 3′-or 5′-UTR. Arrowhead marks the location of gRNA. Arrows show the position of primers for qRT-PCR. Middle panel shows the sequence around gRNA target in wild-types and mutants. The gRNA target sequence is underlined. The insertion nucleotides are marked with red background. Bottom panel shows the truncated Mucin-5AC protein predicted according to the DNA insertion. (B) qRT-PCR analysis of muc5AC gene in muc5AC mutant and wild-type larvae at 5 dpf. (C,D) Representative images of wild-type and muc5AC mutant zebrafish at 3 months post-fertilization (mpf). Scale bar: 5 mm. (E,F) Representative images of cupulae in wild-type and mutant larvae at 5 dpf. Scale bar: 50 μm. (G) Quantification of cupula length in wild-type and muc5AC mutant larvae at 5 dpf. (H) Quantification of cupula diameter in wild-type and muc5AC mutant larvae at 5 dpf. ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Deficiency of muc5AC increases the startle response latency. (A) Schematic diagram of the setup for vibration-induced startle response assay. The vibration module in Zebrabox is set to 70% maximum intensity and a frequency of 400 Hz. A high-speed camera is used to detect the movement of larvae. (B) Schematic diagram of the startle response experiment. T = 0 is the time when vibration stimuli is delivered. (C) Stages of the startle response of wild-type and muc5AC mutant larvae at 5 dpf. T = 0 represents stimulus onset. Arrowheads point to the detectable movement. Dashed rectangles highlight the whole process of startle response. T, ms. (D) Quantification of the short-latency startle response incidence rate in wild-type and muc5AC mutant larvae at 5 dpf. (E) Quantification of the short-latency startle response time in wild-type and muc5AC mutant larvae at 5 dpf. (F) Quantification of the startle response duration time in wild-type and muc5AC mutant larvae at 5 dpf. ***, p < 0.001; ns, not significant.

Schematic diagram of the cupula in wild-type and muc5AC mutant neuromasts. Mucin-5AC is synthesized in support cells and assembled into polymer hydrogels around the cilium bundle.