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Figure 1 - Supplemental 1 Production and characterization of the rpgrip1? and rpgrip1lex4 fish lines. (A) Schematic exon-intron structure of the Danio rerio rpgrip1l gene. Two alleles were produced by CRISPR/cas9-mediated genome engineering, rpgrip1lex4 with a nonsense mutation in exon 4 and rpgrip1l?4-25 (also called rpgrip1l? or rpgrip1l-) with a large deletion between exons 4 and 25, encompassing most of the protein coding sequence. Both alleles had identical embryonic phenotype (panel B and data not shown). (A?) Electrophoresis gel showing the migration profile of amplicons for each genotype, rpgrip1l?/?, rpgrip1l?/+, rpgrip1+l/+ produced by triplex PCR (B) Absence of laterality defects and kidney cysts in rpgrip1l?/? and rpgrip1lex4/ex4 embryos. (C) Immunostaining on whole 30 hpf control (left panels) and rpgrip1l-/- (right panels) embryos to visualize the RF (anti-RF antibody, middle panels) and cilia (acetylated tubulin antibody, lower panels) in the caudal region. Nuclei (upper panels) are stained with DAPI. The RF is present in the central canal of rpgrip1l-/- embryos, as well as cilia in the whole neural tube width. Scale bar: 6 µm for upper panels; 15 µm for lower panels. (D, E) Absence of retinal morphological defects in 5 dpf rpgrip1l-/- larvae. Retinal sections of control (D) and rpgrip1l-/- (E) larvae were immunostained with Zpr3 (red) to label the external segment of photoreceptors and Acetylated Tubulin (Ac Tub) (green) to label axonemes. (D?) and (E?) are insets in (D) and (E) focusing on the photoreceptor layer. Scale bar: 50 ?m. (F-H???) Scanning electron microscopy of brain ventricles in control (F-F??), straight (str.) rpgrip1l-/- (G-G??) and scoliotic (sco.) rpgrip1l-/- (H-H???) adult (3 months) fish. Ependymal multiciliated cells of the rhombencephalic ventricle (RhV) are almost totally absent in rpgrip1l-/- scoliotic fish (H-H'), while they are present in straight rpgrip1l-/-(G-G') and controls (F-F'). Cilia of hindbrain mono-ciliated cells in rpgrip1l-/- (F??, G??) and controls (G??). Scale bars: 10 ?m for (F-I), 2 ?m for F?-H?, 1 ?m for F??, G??, H?? and H???.