Rbm24a is expressed in the otic vesicles and lateral line neuromasts of the zebrafish. Expression pattern of rbm24a during development was examined by performing in situ hybridization of zebrafish embryos at 36 hpf (A–A”), 48 hpf (B–B”), 72 hpf (C,C’), and 96 hpf (D,D’). Otic vesicles are indicated with arrows, and lateral line neuromasts are indicated with arrowheads. Scale bars, 0.25 mm in (A–D), 0.1 mm in (A’–D’) and (A”,B”).

Hair cell function is affected in rbm24a mutants. (A) The auditory function of zebrafish larvae at 5 dpf is evaluated by examining the startle response to various sound intensity. ***p < 0.001. (B) The vestibular function of zebrafish larvae at 5 dpf is evaluated by vestibular head tilt response measurement. ***p < 0.001. (C) The function of hair cells is evaluated by performing FM1-43FX uptake experiment at 3 dpf. Scale bar, 0.5 mm. (D) The numbers of labeled pLL neuromasts per larvae are calculated according to the results from (C). P value < 0.001. The numbers of larvae for each group are indicated in brackets.

Otolith formation and semicircular canal fusion are delayed in rbm24a mutants. (A) Otolith formation in zebrafish larvae is examined using a light microscope at 24, 36, and 48 hpf as indicated. Otoliths are indicated with arrows. Scale bar, 0.1 mm. (B) The numbers of otic vesicles per larvae at 24 hpf are calculated according to the results from (A). P values are 0.000833 (larvae with 4 otoliths), 0.020678 (larvae with 5 otoliths), and 0.00044 (larvae with 6 otoliths). (C) Fusion of the semicircular canals is examined using a light microscope at 50, 60, and 72 hpf as indicated. Scale bar, 50 μm. (D) The numbers of larvae with normal or delayed semicircular canal fusion are calculated according to the results from (C). P values are 0.000001 (50 hpf), 0.00005 (60 hpf), and 0.000034 (72 hpf). The numbers of larvae for each genotype are indicated in brackets. A bulge, anterior bulge; A proj., anterior projection; P bulge, posterior bulge; P proj., posterior projection; V bulge, ventral bulge; V proj., ventral projection; and V pillar, ventral pillar.

Early inner ear development is largely unaffected in rbm24a mutants. Inner ear development is examined by performing in situ hybridization of dlx3(A,B), pax2a(C), myo7aa(D), and dfna5a(E) at different developmental stages as indicated. (F) The numbers of myo7aa⁺ neuromasts are calculated according to the results from (D). (G) The numbers of dfna5a⁺ neuromasts are calculated according to the results from (E). The numbers of larvae for each group are indicated in brackets. Scale bars, 0.2 mm. **p < 0.01; ***p < 0.001.

Development of pLL hair cells is affected in rbm24a mutants. (A) Confocal microscopic imaging analysis of pLL neuromasts in wild-type or rbm24a mutant brn3c:GFP line at different developmental stages as indicated. (B,C) The numbers of GFP-positive hair cells of each pLL neuromasts at 48 and 96 hpf are calculated according to the results from (A). The numbers of larvae for each group is 15. (D,D’) Hair bundles of pLL neuromasts at 72 hpf are examined using SEM. Stereocilia are indicated with arrows. Scale bars, 10 μm in (A), 2 μm in (D,D’). ns, not significant; *p < 0.05; and ***p < 0.001.

Development of lateral crista hair cells is affected in rbm24a mutants. Confocal microscopic imaging analysis of rbm24a mutant brn3c:GFP line at 72 hpf (A) and 96 hpf (D). (B,E) Stereocilia length is calculated according to the results from (A,D), respectively. (C,F) Kinocilia length is calculated according to the results from (A,D), respectively. For stereocilia length measurement at 72 hpf, brn3c:GFP, n = 21 (from 3 larvae); brn3c:GFP;rbm24a^{– /–}, n = 20 (from 4 larvae). For stereocilia length measurement at 96 hpf, brn3c:GFP, n = 20 (from 3 larvae); brn3c:GFP;rbm24a^{– /–}, n = 25 (from 4 larvae). For kinocilia length measurement at 72 hpf, brn3c:GFP, n = 20 (from 3 larvae); brn3c:GFP;rbm24a^{– /–}, n = 20 (from 4 larvae). For kinocilia length measurement at 96 hpf, brn3c:GFP, n = 30 (from 3 larvae); brn3c:GFP;rbm24a^{– /–}, n = 26 (from 3 larvae). Scale bar, 10 μm. ***p < 0.001.

Dysregulated genes in the inner ear of rbm24a mutants. (A) Dysregulated genes in the inner ear of rbm24a mutants identified from RNAseq analysis. RT-PCR (B) and qPCR (C) were performed to confirm the RNAseq results. Random primers or oligo dT were used as primers for reverse transcription as indicated. (D) RIP experiments were performed to examine the binding of Rbm24a with its target mRNAs. Statistical analysis of the qPCR results shows that the expression difference is significant (P value < 0.000001, except that P value of cyp2aa7 is 0.001315).

Expression pattern of important Rbm24a target mRNAs in the zebrafish. In situ hybridization of zebrafish embryos at 34 hpf was performed to examine the expression of dfna5b, epcam, casp6a, lrrc23, enosf1, dnah7, and smpx. (A) Lower magnification images. (B) Higher magnification images. Scale bars, 0.25 mm in (A) and 0.1 mm in (B).