Journal: medRxiv

Article Title: FRMPD4 , a causal gene for intellectual disability and epilepsy, is associated with X-linked non-syndromic hearing loss

doi: 10.64898/2026.03.27.26349271

Figure Lengend Snippet: Loss of function of frmpd4 in zebrafish results in neuromast alterations in the otic vesicle and in the posterior lateral line. (A) Functional test of frmpd4 splice blocking Morpholino via RT-PCR (per condition 10 pooled embryos). Exon-spanning PCR primers indicate the loss of a correctly spliced frmpd4 band after Morpholino injection, and gain of a larger, unspliced additional PCR product. PCR amplification of ef1a1 were used as internal cDNA control. (B) DASPEI staining for neuromasts in frmpd4 Morpholino injected knockdown embryos. Quantification of neuromast number in the posterior lateral line (C) , the otic vesicle (D) and the preoptical/supraorbital (E) indicated reduction after frmpd4 knockdown (0.25mM Morpholino concentration, age: 4 dpf, comparison to embryos injected with standard MO). (F) Measurement of neuromast distance in the posterior lateral line indicated no significant change after frmpd4 Morpholino knockdown. (G) Staining for acetyl tubulin after frmpd4 Morpholino knockdown in 4 dpf larvae indicated similar to in frmpd4 sa12377 mutants loss of neuronal cells and axonal projections in ventral sensory patches of the otic vesicle and interference with correct neuromast formation in the posterior lateral line. Analyzed embryos: 2 wild type controls; 4 frmpd4 MO 0.25mM; 2 frmpd4 MO 0.5mM.

Article Snippet: Afterwards, the tissue was incubated with a primary antibody solution containing 3% normal horse serum, 1% bovine serum albumin, 0.3% Triton X-100, and 0.1% Tween20 with primary antibodies for βII-tubulin (mouse monoclonal, R&D System, #MAB1192 clone Tuj1, dilution 1:1000) and FRMPD4 (rabbit, polyclonal, dilution 1:500) ( ) incubated overnight at 4°C.

Techniques: Functional Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Injection, Amplification, Control, Staining, Knockdown, Concentration Assay, Comparison

Journal: medRxiv

Article Title: FRMPD4 , a causal gene for intellectual disability and epilepsy, is associated with X-linked non-syndromic hearing loss

doi: 10.64898/2026.03.27.26349271

Figure Lengend Snippet: frmpd4 CRISPant in zebrafish show only mild neuromast alterations in the otic vesicle and in the posterior lateral line. (A) DASPEI staining for neuromasts in CRISPants embryos (F0 generation, frmpd4 sgRNA r2 and f3 injected). Quantification of neuromast number in the posterior lateral line (B) , the otic vesicle (C) , and the preoptical/supraorbital (D) indicated reduction in frmpd4 CRISPants (4 dpf). (E) Measurement of neuromast distance in the posterior lateral line indicated no significant change after frmpd4 CRISPR or Morpholino knockdown. (F) Staining for acetylated tubulin in 4 dpf embryos indicated mild changes to neuronal cells and axonal projections in ventral sensory patches of the otic vesicle and not significant interference with correct neuromast spacing in the posterior lateral line after transient frmpd4 CRISPR knockdown. Analyzed embryos: 2 wild type controls; 4 frmpd4 CRISPants.

Article Snippet: Afterwards, the tissue was incubated with a primary antibody solution containing 3% normal horse serum, 1% bovine serum albumin, 0.3% Triton X-100, and 0.1% Tween20 with primary antibodies for βII-tubulin (mouse monoclonal, R&D System, #MAB1192 clone Tuj1, dilution 1:1000) and FRMPD4 (rabbit, polyclonal, dilution 1:500) ( ) incubated overnight at 4°C.

Techniques: Staining, Injection, CRISPR, Knockdown

Journal: medRxiv

Article Title: FRMPD4 , a causal gene for intellectual disability and epilepsy, is associated with X-linked non-syndromic hearing loss

doi: 10.64898/2026.03.27.26349271

Figure Lengend Snippet: Loss of function of frmpd4 in zebrafish results in axonal and structural malformations in the otic vesicle and the posterior lateral line. (A) Staining for acetylated tubulin in 4 dpf embryos indicated loss of neuronal cell and axonal projection in ventral sensory patches of the otic vesicle in homozygous frmpd4 sa12377/sa12377 mutants. (B) Posterior lateral line neuromasts and axons (white arrowheads) are affected by frmpd4 loss by depicting size reduction and morphological changes (analyzed embryos per genotype: 7 wild type controls; 6 heterozygous frmpd4 sa12377/+ ; 5 homozygous frmpd4 sa12377/sa12377 ). (C) Neuromast cell deposition in the PLL can be disrupted in homozygous frmpd4 sa12377/sa12377 mutants, while adjacent somites show normal patterns (n=3 embryos per genotype; control and heterozygous frmpd4 sa12377/+ mutants display indistinguishable patterns). (D) Scanning electron microscopy further showed disruption of cellular organization in PLL neuromasts and loss of kinocilia in in frmpd4 sa12377/sa12377 mutants (n=3 per genotype). m: anterior macula, pm: posterior macula, ac: anterior crista, lc: lateral crista, pc: posterior crista. Scale bars in A to C indicate 50 µm.

Article Snippet: Afterwards, the tissue was incubated with a primary antibody solution containing 3% normal horse serum, 1% bovine serum albumin, 0.3% Triton X-100, and 0.1% Tween20 with primary antibodies for βII-tubulin (mouse monoclonal, R&D System, #MAB1192 clone Tuj1, dilution 1:1000) and FRMPD4 (rabbit, polyclonal, dilution 1:500) ( ) incubated overnight at 4°C.

Techniques: Staining, Control, Electron Microscopy, Disruption