The ndrg2 gene is conserved across multiple species and highly expressed in early developmental otic vesicles and neuromasts in posterior lateral line (pLL). (A) Amino acid sequence alignment of zebrafish (Danio rerio) ndrg2 (marked with a red star), human ndrg2 and homologous genes in several mammalian animals. (B) The established phylogenetic tree utilizing Neighbor-Joining method and MEGA software (version 6.0) containing zebrafish ndrg2 (marked with a red star), human ndrg2 and its orthologs in other species. (C–E) Expression profiles of ndrg2 mRNA examined via whole-mount in situ hybridization (WISH) with the lateral view in zebrafish embryos at 24, 48 and 72 hpf, respectively. The positive signals of ndrg2 mRNA were focused on the otic vesicle (marked with red dashed box) and neuromasts in pLL (marked with red arrowheads) during early embryo development. A schematic of the inner ear structure in zebrafish was shown in (D). UO, utricular otolith; SO, saccular otolith; UM, utricular macula; SM, saccular macula; AC, anterior crista; LC, lateral crista; PC, posterior crista. (C’,D’,E’) Magnified images of the positive signals in otic vesicle with lateral view in zebrafish at 24, 48 and 72 hpf, respectively. (C’’) A dorsal view of otic vesicles in zebrafish at 24 hpf. (D’’,E’’) Enlarged images of the positive signals in neuromasts in pLL (marked with red arrowheads) with lateral view in zebrafish at 48 and 72 hpf, respectively.

Knockdown of the ndrg2 gene led to significantly reduced crista hair cells (HCs), shortened kinocilia, decreased neuromasts as well as reductive functional HCs. (A) Schematic structure of main auditory organs in zebrafish (Danio rerio) at 72 hpf, including otic vesicle and posterior lateral line (pLL) system. Detailed structures of both otic vesicle and crista HCs cluster were depicted and the typical three clusters of crista HCs comprised anterior crista hair cell (ACHC), lateral crista hair cell (LCHC) and posterior crista hair cell (PCHC). The neuromasts in pLL that were analyzed in this study were marked with red dashed box. (B) Representative fluorescence images of HC clusters (marked with red arrowheads) in pLL at 72 hpf in the control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively. Scale bars: 500 µm. (C) Lateral views of eya1 mRNA expression detected via whole-mount in situ hybridization (WISH) in normal, ndrg2 morphants, and ndrg2 mRNA rescued larvae at 72 hpf. The positive signals in neuromasts in the pLL were pointed with red arrowheads. (D) Representative fluorescence images of the three clusters of crista HCs at 72 hpf in control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively. The details of LCHC (marked with white dashed box) were enlarged in corresponding groups. Scale bars: 20 µm. (E) Representative enlarged micrographs of HC cluster (green color) and functional HC cluster (red color) in single neuromast in pLL at 72 hpf in control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively. Scale bars: 10 µm. (F–H) Statistical analysis of the number of crista HCs, the mean length of kinocilia and stereocilia at 72 hpf in the control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively ((F,G) n = 16; (H), n = 10). (I) Statistical analysis of the number of HCs and functional HCs per neuromast at 72 hpf in control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively (n = 48). (J,K) Statistical analysis of the number of HC clusters and neuromasts in pLL in control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively ((I), n = 16; (J), n = 13). Symbols of * and **** above bars represent p < 0.05 and p < 0.0001, respectively. ns, no significance.

Knockdown of the ndrg2 gene induced weakened response behavior to sound stimuli in zebrafish (Danio rerio) at 5 dpf. (A) Schematic diagram of the devices used in startle response assay and sound vibration stimuli modes applied to larvae. (B) Extracted locomotion trajectories from larvae with C-shape motion under one-time stimulus of 9 dB re.1 ms⁻² sound level with 180 Hz tone bursts in control, ndrg2 morphants, and ndrg2 mRNA rescued groups, respectively. (C,D) Statistical analysis of the mean distance and peak velocity of locomotion under two levels stimuli in normal, ndrg2 morphants, and ndrg2 mRNA rescued larvae at 5 dpf, respectively (n = 20). Symbols of *, **, ***, and **** above bars represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. ns, no significance.

CRISPR/Cas9-mediated ndrg2 mutation was successfully generated at the target site. (A) Schematic diagram of zebrafish (Danio rerio) ndrg2 genomic structure and ndrg2-specific sgRNA targeting exon 2 in the CRISPR/Cas9 system. (B) Multiple mutations occurred at the target site in ndrg2 mutants in comparison with wild-type zebrafish.

Knockout of the ndrg2 gene disrupted hair cell (HC) morphogenesis and caused defective sensory ability to sound vibration stimuli in zebrafish (Danio rerio). (A) Representative fluorescence graphs of the typical three clusters of crista HCs in normal, ndrg2 mutants, and ndrg2 mRNA rescued larvae at 72 hpf, respectively. The details of lateral crista hair cell (LCHC) (marked with white dashed box) were magnified in corresponding larvae. Scale bars: 20 µm. (B) Phase-contrast and fluorescence images of HC clusters (marked with red arrowheads) in posterior lateral line (pLL) at 72 hpf from the control, ndrg2 mutants, and ndrg2 mRNA rescued groups, respectively. Scale bars: 500 µm. (C) Overlapped fluorescence images of a representative neuromast in pLL from normal, ndrg2 mutants, and ndrg2 mRNA rescued larvae at 72 hpf, respectively. The green and red signals represented HCs and functional HCs, respectively. Scale bars: 10 µm. (D) Swimming trajectories extracted from once startle response behavior to sound stimuli of 9 dB re.1 ms⁻² with 180 Hz tone bursts in the normal, ndrg2 mutants, and ndrg2 mRNA rescued larvae at 5 dpf, respectively. (E–I) Statistical analysis of the morphological phenotypes of HCs emerging in ampulla crista of otic vesicle and neuromasts in pLL at 72 hpf in the control, ndrg2 mutants, and ndrg2 mRNA rescued groups, respectively ((E–H), n = 10; (I), n = 30). (J,K) Statistical analysis of the mean distance and peak velocity of locomotion under two levels stimuli in the control, ndrg2 mutants, and ndrg2 mRNA rescued groups, respectively (n = 20). Symbols of *, **, ***, and **** above bars represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. ns, no significance.

Loss of the ndrg2 gene in Tg(Brn3c:mGFP) zebrafish (Danio rerio) influenced hair cells’ (HCs’) differentiation through Notch signaling without changes in HCs’ apoptosis and supporting cells. (A) Results of HC apoptosis assay in normal and ndrg2 mutant larvae at 72 hpf. Immunofluorescence images were shown to recognize the nucleus (blue color), HC (green color), and cleaved caspase-3 signal (red color) in a single HC cluster in posterior lateral line (pLL). Scale bars: 10 µm. (B) Results of supporting cell proliferation assay in normal and ndrg2 mutant larvae. Immunofluorescence micrographs were displayed to identify the HC (green color), supporting cell (red color) and proliferating cell (blue color) in a single neuromast in pLL at 96 hpf. Alexa Fluor^TM 647 used to detect BrdU was painted in pseudo blue color to distinguish from the red color of Alexa Fluor^TM 555 labeling SOX2. The purplish-red signals in the last column of overlapped fluorescence images represented the proliferating supporting cells. Scale bars: 10 µm. (C) Representative images of HC cluster (green color) and functional HC cluster (red color) in single neuromast in pLL at 72 hpf in control, ndrg2 mutants and treatment with Notch inhibitor LY411575 groups, respectively. Scale bars: 10 µm. (D) Statistical analysis of the number of HCs and functional HCs per neuromast at 72 hpf in control, ndrg2 mutants and treatment with Notch inhibitor LY411575 groups, respectively (n = 30). (E,F) Statistical analysis of the number of supporting cells and proliferating supporting cells per neuromast at 96 hpf in normal and ndrg2 mutant larvae (n = 30). Symbols of **** above bars represent p < 0.0001. ns, no significance.