Fig. 4

Transient suppression or CRISPR/Cas9 mediated genome-editing of cep55l results in craniofacial, neurological and renal abnormalities in zebrafish. (A) Representative live ventral views of 4 dpf -1.4col1a1:eGFP larvae showing craniofacial structures. cep55l F0 mutants or sb2 morphant larvae show significantly broader ceratohyal (CH) angles compared with controls. (B) Plot indicates the measurement of the ceratohyal angle (as shown in panel A). RNA injected alone shows no effect. Larvae injected with sb2 or sgRNA+Cas9 display significantly increased ceratohyal angle compared with controls. This phenotype can be rescued with coinjection of sb2 and wild-type (WT) (p.H378-RNA) or p.L378-RNA. sb2+S425X RNA does not rescue significantly. n=23–62 embryos/injection; repeated. (C) 4 dpf larvae stained for acetylated tubulin show hypoplasia of brain regions, including the cerebellum and optic tecta. Box and zoomed image indicate the cerebellum; asterisks (*) indicate optic tecta. (D) Plot indicates the quantification of the total area (µm²) of the cerebellum at 4 dpf, as indicated by orange outline in the control and sb2 insets of (C). n=31–81 embryos/injection; repeated. (E) Larvae fixed at 4 dpf and stained for Na⁺/K⁺ATPase are shown (lateral view; zoomed image represents the proximal convoluted tubule; outline indicates the area quantified in (F). Embryos injected with cep55 sb2 or sgRNA + Cas9 display proximal tubules of decreased area (µm²) and decreased presence of convolutions compared with control embryos. (F) Plot indicates the total area (µm²) of the proximal tubule in 4 dpf larvae; n=31–61 embryos/injection, repeated. *, **, *** and **** indicate p<0.05, 0.01, 0.001 and 0.0001 respectively. ns, not significant.