High expression of HSD17B7 in zebrafish and mice sensory hair cells

(A) The UMAP analysis of the zebrafish scRNA-seq data. 0, 2, 3, and 5 clusters of cells were annotated as hair cells. 0, neuromast hair cell; 1, supporting cell; 2, macula hair cell; 3, crista hair cell 1; 4, mantle cell; 5, crista hair cell 2. (B) and (C) Feature plot and violin plot of hsd17b7. (D) hsd17b7 expression increased along the pseudotime trajectory of neuromast hair cell formation. (E) Increase in hsd17b7 gene expression levels along the hair cell trajectory. (F) Heatmap of marker genes along the pseudotime trajectory. (G) The expression of the hsd17b7 in 72 hpf and 96 hpf embryos was detected by whole-mount in situ hybridization analysis. Dashed circles indicate the otic vesicle and the neuromast. (H) and (I) Representative images of the crista hair cells and neuromast hair cells in Tg(myo6b: hsd17b7-egfp) at 4 dpf. NM hair cells, neuromast hair cells. Scale bars, 10 μm. (J) The average expression of Hsd17b7 and hair cell marker genes across mouse hair cell types. (K) Immunostaining analysis of HSD17B7 expression in mouse cochlea hair cells at P60. MYO7A was used as a marker for hair cells. The dashed box indicates the magnified region. OHC, outer hair cell; IHC, inner hair cell.

Knockout of hsd17b7 caused compromised MET function and hearing defects in zebrafish

(A) Left: Schematic diagram of the devices used in the startle response assay applied to larvae. Right: Extracted locomotion trajectories from larvae with C-shape motion under a one-time stimulus of 9 dB re.1 ms-2 sound level with 60 Hz tone bursts in control, hsd17b7 KO, and mRNA rescued groups, respectively. Scale bars, 10 mm. (B) and (C) Quantification of the peak velocity and mean distance of movement at 5 dpf larvae under sound stimuli for (A) (n=20). (D) Representative images of neuromast hair cells (HCs, green) in Tg(Brn3C: mGFP) at 4 dpf larvae from control, hsd17b7 KO, and hsd17b7 mRNA rescued groups. Scale bars, 20 μm. (E) Quantification of the number of HCs per neuromast for (D) (n=30). (F) Representative images of neuromast HCs (green) and functional HCs (red) in single neuromast of Tg(Brn3C: mGFP) at 5 dpf larvae from control, hsd17b7 KO, and hsd17b7 mRNA rescued groups, respectively. The white dashed circles indicate NM HCs. Scale bars, 20 μm. (G) Quantification of the FM4-64 relative intensity of HCs per neuromast for (F) (n=27). All quantification data (B, C, E, and G) are presented as the mean ± SD. P values were determined using a one-way ANOVA test followed by Tukey’s multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant, p > 0.05.

HSD17B7 deficiency reduced cholesterol in hair cells

(A) Schematic representation of the cholesterol synthesis pathway. (B) Western blots showing HSD17B7 protein levels in HSD17B7 Knock-down HEI-OC1 cells. Quantification of relative protein levels of HSD17B7 is shown on the right (n = 6). (C) Quantification of the relative cholesterol levels in HSD17B7 knock-down HEI-OC1 cells (n=12). (D) Immunostaining shows cholesterol probe D4H-mCherry in HEI-OC1 cells transfected with pCMV2-Flag and pCMV2-Flag-HSD17B7 full-length plasmids, respectively. Scale bars, 20 μm. (E) Quantification of D4H relative intensity in vitro for (D) (n=16). (F) Schematic diagram of hsd17b7 KO one-cell embryos injection of pmyo6b-D4H-mCherry in Tg(Brn3C: mGFP). (G) Representative images of cholesterol probe D4H-mCherry (red) expression in crista HCs (left) and NM HCs (right) of control or hsd17b7 KO mutant larvae at 4 dpf. Scale bars, 20 μm. (H) Quantification of D4H relative intensity in vivo for (G) (n=30). All quantification data (B, C, E, and H) are presented as the mean ± SD. P values were determined using a two-tailed unpaired Student’s t-test. ***P < 0.001; ****P < 0.0001.

Identification of a novel heterozygote nonsense mutation in HSD17B7

(A) Two-generation family pedigree for the affected individual. The hearing-impaired individual is indicated by a black cycle (female). (B) Pure-tone audiometry audiograms for the hearing-impaired individual at 8 years old. Blue represents the results for the left ear and red for the right ear. Affected individual shows severe-to-profound or profound HI. (C) Sequence of HSD17B7 mRNA in Wild type and Heterozygote. (D) Multiple sequence alignment of 8 different species using TBtools program. The p.Glu182 residue as indicated by a red arrow is evolutionarily conserved from zebrafish to human. (E) The domain structure of human HSD17B7WT and HSD17B7E182*. ED, extracellular domain; TM, transmembrane; CD, cytoplasmic domain. The residue numbers are labeled at right. (F) SWISS-MODEL predicts the three-dimensional protein structure of HSD17B7WT and HSD17B7E182*.

E182* mutation mRNA of HSD17B7 failed to rescue the impaired MET function and auditory behavior of hsd17b7 mutants

(A) Representative images of NM HCs (green) and functional NM HCs (red) in single neuromast of Tg(Brn3C: mGFP) at 5 dpf larvae from control, hsd17b7 KO, KO + E182* mRNA and KO + WT mRNA groups. White dashed circles indicate NM HCs. Scale bars, 20 μm. (B) Quantification of the FM4-64 relative intensity of HCs per neuromast for (A) (n=25). (C) Extracted locomotion trajectories from 5 dpf larvae with C-shape motion under a one-time stimulus of 9 dB re.1 ms-2 sound level with 60 Hz tone bursts in control, hsd17b7 KO, KO + E182* mRNA, and KO + WT mRNA groups, respectively. Scale bars, 10 mm. (D) and (E) Quantification of the mean distance and peak velocity of movement at 5 dpf larvae under sound stimuli for (C) (n=20). All quantification data (B, D, and E) are presented as the mean ± SD. P values were determined using a one-way ANOVA test followed by Tukey’s multiple comparisons. ****P < 0.0001; ns, non-significant, p > 0.05.

Subcellular localization of HSD17B7 WT and E182* mutation

(A) Immunostaining shows the subcellular localization of Flag-HSD17B7WT and Flag-HSD17B7E182* in HEI-OC1 cells. Calnexin was used as a marker for ER. The white arrow indicates the profile position in (B, and C). Scale bars, 20 μm. (B) and (C) The intensity profile shows the localization of HSD17B7WT and HSD17B7E182* with ER, respectively. (D) Immunostaining shows the subcellular localization of HSD17B7WT and HSD17B7E182* in one HEI-OC1 cell. The white arrow indicates the profile position in (E). Scale bars, 20 μm. (E) The intensity profile shows the localization of HSD17B7WT and HSD17B7E182* in one HEI-OC1 cell.

The heterozygous E182* mutation leads to a negative effect on auditory function

(A) Representative images of neuromast HCs (green) and functional HCs (red) in single neuromast of Tg(Brn3C: mGFP) at 5 dpf larvae from control, HSD17B7E182* mRNA and HSD17B7WT mRNA groups, respectively. White dashed indicate NM HCs. Scale bars, 20 μm. (B) Quantification of the FM4-64 relative intensity of HCs per neuromast for (A) (n=34). (C) Extracted locomotion trajectories from larvae with C-shape motion under a one-time stimulus of 9 dB re.1 ms-2 sound level with 60 Hz tone bursts in control, HSD17B7E182* mRNA and HSD17B7WT mRNA groups, respectively. Scale bars, 10 mm. (D) and (E) Quantification of the mean distance and peak velocity of movement at 5 dpf larvae under sound stimuli for (C) (n=20). (F) Immunostaining shows the distribution of cholesterol probe D4H in HEI-OC1 cells transfected with pCMV2-Flag, pCMV2-Flag-HSD17B7WT, and pCMV2-Flag-HSD17B7E182* plasmids, respectively. The white arrow indicates the profile position in (G, H, and I). Scale bars, 20 μm. (G), (H), and (I) The intensity profile shows the localization of Flag, HSD17B7WT, and HSD17B7E182* with the cholesterol probe D4H, respectively. All quantification data (B, D, and E) are presented as the mean ± SD. P values were determined using a one-way ANOVA test followed by Tukey’s multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, non-significant, p > 0.05.

HSD17B7WT binds with Retention in endoplasmic reticulum 1 (RER1)

(A) Flag, Flag-HSD17B7WT, and Flag-HSD17B7E182* were immunoprecipitated from HEI-OC1 cells transfected with pCMV2-Flag, pCMV2-Flag-HSD17B7WT, and pCMV2-Flag-HSD17B7E182* plasmids, respectively. Tubulin and IgG light chains were used as the loading control. Cell lysates were immunoprecipitated with anti-Flag beads. (B) The Venn diagram shows the number of HSD17B7WT interacting proteins (red) and HSD17B7E182* interacting proteins (blue) identified in LC-MS/MS. The number of proteins in each area is marked. (C) Biological Process GO term enrichment analysis for the HSD17B7WT-specific interacting proteins and HSD17B7E182*-specific interacting proteins identified in LC-MS/MS. (D) Cellular Component GO term enrichment analysis for the HSD17B7WT-specific interacting proteins and HSD17B7E182*-specific interacting proteins identified in LC-MS/MS. (E) The top 20 (iBAQ intensity) for the HSD17B7WT-specific interacting proteins in the ER. (F) In vitro binding assays show the interaction between HSD17B7 and RER1. Top: GST and GST-HSD17B7 used for the pull-down assay were stained with Coomassie blue. Bottom: Western blot for RER1-His. (G) Immunostaining shows the localization of RER1-Myc in HEI-OC1 cells transfected with pCMV2-Flag-HSD17B7WT and pCMV2-Flag-HSD17B7E182* plasmids, respectively. The white arrow indicates the profile position in (H, and I). Scale bars, 20 μm. (H) and (I) The intensity profile shows the localization of HSD17B7WT and HSD17B7E182* with RER1, respectively.

Evolutionary conservation of HSD17B7

(A) Evolutionary conservation of HSD17B7. The phylogenetic tree was reconstructed in the Phylogeny.fr platform (v3.1/3.0 aLRT) based on the maximum likelihood method, which graphical representation and edition were performed with TreeDyn (v198.3). (B) The amino acid sequence was analyzed by the TBtools multiple sequence alignment of HSD17B7 orthologs.

Generation of hsd17b7 mutant using CRISPR/Cas9 system in zebrafish

(A) Schematic diagram showing sgRNA and the target site on the exon 3 of hsd17b7 gene. (B) Schematic diagram showing the hsd17b7 mutant establishment process. (C) Mutation pattern of sgRNA and Cas9-injecting embryos. (D) Sequencing Chromatograms of −4 bp, −6 bp and −6 bp deletion mutant line (E) Three types of mutations were identified by sequencing and screening. (F) Schematic diagram of the predicted proteins encoded by −4 bp deletion mutant line.

Knockdown of hsd17b7 leads to auditory dysfunction in zebrafish

(A) Schematic diagram of morpholino and its target site at the exon 1/intron 1 junction of hsd17b7 gene. (B) Schematic diagram of single-cell embryo injection of hsd17b7 morpholino (Mo) in wild-type strain (AB). (C) The efficiency of morpholino was detected by RT-PCR. (D) Western blots showing Hsd17b7 protein levels of control, hsd17b7 morphants, and hsd17b7 mRNA rescued groups in whole zebrafish (3 dpf). Quantification of relative protein levels of Hsd17b7 is shown on the right (n=7). (E) Extracted locomotion trajectories from larvae with C-shape motion under a one-time stimulus of 9 dB re.1 ms-2 sound level with 60 Hz tone bursts in control, hsd17b7 morphants, and hsd17b7 mRNA rescued groups, respectively. Scale bars, 10 mm. (F) and (G) Quantification of the mean distance and peak velocity of movement at 5 dpf larvae under sound stimuli for (E) (n=20). All quantification data (D, F, and G) are presented as the mean ± SD. P values were determined using a one-way ANOVA test followed by Tukey’s multiple comparisons. **P < 0.01; ns, non-significant, p > 0.05.

Knockdown of hsd17b7 leads to MET dysfunction and decreases cholesterol levels in zebrafish

(A) Representative images of neuromast HCs (green) and functional HCs (red) in single neuromast of Tg(Brn3C: mGFP) at 5 dpf larvae from control, hsd17b7 morphants, and hsd17b7 mRNA rescued groups, respectively. White dashed indicated neuromast HCs. Scale bars, 20 μm. (B) Quantification of the FM4-64 relative intensity of HCs per neuromast for (A) (n=23). (C) and (D) Representative images of cholesterol probe D4H-mCherry (red) expression in crista HCs (green) and neuromast HCs (green) of control or hsd17b7 morphants at 4 dpf. Scale bars, 20 μm. (E) and (F) Quantification of D4H relative intensity in crista HCs and neuromast HCs for (C, and D) (n=24). Quantification data (B) are presented as the mean ± SD. P values were determined using a one-way ANOVA test followed by Tukey’s multiple comparisons. ****P < 0.0001; ns, non-significant, p > 0.05. Quantification data (E, and F) are presented as the mean ± SD. P values were determined using a two-tailed unpaired Student’s t-test. ****P < 0.0001.

Schematic of the experimental process for immunoprecipitation and LC-MS/MS to identify HSD17B7WT and HSD17B7E182* interacted proteins.

E182* mutation decreased HSD17B7 protein levels, mRNA levels and the half-life periods

(A) The phenomena of transfecting with pCMV2-Flag, pCMV2-Flag-HSD17B7WT, and pCMV2-Flag-HSD17B7E182* plasmids in HEI-OC1 cells. (B) Western blots showing the protein levels of exogenously expressed HSD17B7 in HEI-OC1 cells transfected with pCMV2-Flag-HSD17B7WT, and pCMV2-Flag-HSD17B7E182* plasmids, respectively. Tubulin was used as the loading control. Red stars indicated HSD17B7WT and HSD17B7E182*. Quantification of relative protein levels of HSD17B7WT and HSD17B7E182*are shown on the right (n=6). (C) RT-qPCR showing the mRNA level of HSD17B7WT and HSD17B7E182* in transfected HEI-OC1 cells (n=11). (D) The stability of mRNA was detected by RT-qPCR in transfected HEI-OC1 cells. (E) Schematic of the process for mRNA stability detection’s experimental setup and sample collection. All quantification data (B, and C) are presented as the mean ± SD. P values were determined using a two-tailed unpaired Student’s t-test. ****P < 0.0001.