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.
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