Phenotypes of auditory system in mfsd3 zebrafish morphants. (A) Schematic of two MO targeting strategies: ATG-MO blocks translation initiation while E2I2-MO disrupts exon 2-intron 2 splicing. Primers pairs (1F/4R) detect wild-type transcripts or aberrant splicing products containing retained intron 2 or skipped exon 2. (B) Left: RT-PCR analysis of mfsd3 transcript from 1 dpf embryos injected with control-MO or E2I2-MO (4 ng). Aberrant splicing product (intron 2 retention and exon2 skipping) are indicated by shifted band patterns. Right: Sanger sequencing confirmation of wild-type sequence, intron 2-inserted transcript and exon2-skipped transcript. (C–Q) Gross morphology of head neuromasts and inner ear in Tg (Brn3c: mGFP) embryos at 3-dpf. Compared to control MO, Mfsd3 deficiency led to reduced hair cell numbers in head neuromasts ((G,H), yellow arrowheads), hypoplastic otic vesicle (J,K), malformed otoliths ((J,K), blue arrowheads) and damaged otic vesicle hair cells ((M–N), (P–Q), asterisk). (R–T) Quantitative measurements demonstrated significant reduction in: otic vesicle area (R), otoliths area (S) and mean otolith diameter (T). ***P < 0.001 (n = 10; ANOVA). (U) Quantitative of hair cell numbers in head neuromasts showing significant decrease in morphants. Scale bar, 100 μm; Scatter plot with bars; ***P < 0.001 (n = 10; ANOVA). dpf, days post fertilization.

Morphological defects and hair cells loss in Mfsd3-deficient zebrafish. (A–C) Compared with control MO, the mfsd3 knockdown MO showing significant body shortening (B,C), axis curvature (B,C) and pericardial edema ((B,C), red arrowheads). (D–L) Live imaging of Brn3c: mGFP transgenic embryo at 3-dpf showed GFP expression from RGCs (retinal ganglion cells) and neuromasts (green dots) of the posterior lateral line and head. Zebrafish under control exhibited normal hair cells number (D). Conversely, significantly decreased hair cells of the posterior lateral line were observed in mfsd3 morphants (E,F). The white boxed regions are presented in amplified form in the lower panels (G–L) GFP fluorescence signal alone (G–I) and GFP fluorescence signal merged with bright field image (J–L). Morphometric analyses were utilized to quantify the fluorescence particle signal of hair cells. (M) A time-course plot of survival rates in control vs. mfsd3 morphants for 3 days. (N) The percentage of embryos with development defects in control vs. mfsd3 morphants. (O,P) Quantitative analysis of body length (O) and curvature angle (P) of embryos. (n = 10; ANOVA; ***P < 0.001). (Q) Quantitative analysis of the pericardial area of embryos. (R) Quantitative analysis of the average number of hair cells at posterior lateral line. Scale bar, 100 μm; Scatter plot with bars (n = 10; ANOVA; **P < 0.0001). dpf, days post fertilization.

Locomotor capacity was reduced in mfsd3 zebrafish morphants. (A,B) The digital tracks and heatmap image in larvae from control-MO and mfsd3-MO injected groups at 5-dpf. Each behavioral test consisted of ten larvae each placed into individual wells. (C–F) The statistical analyses showed significantly reduced total movement distance (C), velocity (D), mobility (E) and maximum acceleration (F) in mfsd3 morphants compared to control-MO injected groups (n = 10; ANOVA; ***P < 0.001). dpf, days post fertilization.

Zebrafish with Mfsd3 deficiency exhibited upregulated Wnt/β-catenin signaling activity. (A) Schematic diagram depicting the potential regulatory action of mfsd3 on the Wnt/β-catenin signaling pathway. (B) Wnt/β-catenin signaling pathway was upregulated in mfsd3 morphants. Endogenous dkk1b, wnt8a, wnt9a, lrp5, lrp6, frzb, fzd7a, fzd7b, gsk-3β, axin1, axin2, mycn, lef1, myca, β-catenin and COX2 in control and mfsd3 morphants measured by qRT-PCR assay (n = 6–10 individual embryos). The dkk1b, wnt8a, lrp6, frzb and COX2 were all upregulated significantly in the mfsd3 morphants group. ***P < 0.001; **P < 0.01; *P < 0.05.