Fig. 4

Genetic interaction between ninl and cc2d2a.

(a-d) Partial ninl knockdown enhances the cystic kidney phenotype of cc2d2a mutants. (a-c) Glomerulus and proximal pronephric tubules highlighted in the transgenic line Tg(wt1b-EGFP). (a) Injection of a low dose of ninl atgMO (0.75 ng/nl) causes no cysts in wild-type larvae. (b) cc2d2a-/- larvae display small dilatations of the proximal tubules (arrow) in ~40% of cases. (c) Injection of this low dose of ninl atgMO in the cc2d2a-/- background leads to large dilatations of the proximal tubules and glomerular space (arrow) in 89% of mutants. g glomerulus, p pancreas. (d) Quantification of the glomerular + proximal tubular area displayed as a scatter plot, demonstrating a significant increase in proximal pronephric area in cc2d2a-/- larvae injected with low-dose ninl atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint is an individual fish. (e-g') Immunohistochemistry with anti-opsin antibody (4D2, green) on retinal cryosections of 4dpf cc2d2a-/- uninjected larvae (f-f”) and cc2d2a-/- larvae injected with subphenotypic doses of ninl MO (g'g”'), that cause no mislocalization in wild-type fish (e-e'), demonstrates that partial ninl knockdown increases the mislocalization of opsins (e'-g'). (h) Quantification of the mean intracellular fluorescence displayed as a scatter plot shows significant increase in intracellular fluorescence in cc2d2a-/- larvae injected with low dose of ninl atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint represents the mean intracellular fluorescence from 10 photoreceptors in one individual fish. Cell membrane and outer segments are stained with bodipy (red in e-g). Nuclei are counterstained with DAPI. Scale bars are 100 μm in (a-c) and 4 μm in (e-g'). (i) Pedigree of a consanguineous family with one affected boy (UW48-3) and 4 unaffected siblings. UW48-3 carried a homozygous missense CC2D2A mutation as well as a frameshift mutation in NINL leading to premature truncation. (j) Pedigree of a family where the affected individual (UW36-3) carries the same homozygous CC2D2A mutation as in (i) but no additional rare deleterious variants. (k) Pedigree of a family where the affected individual (UW07-3) carries compound heterozygous C5ORF42 frameshift mutations and a nonsense mutation in NINL. (l) Pedigree of a family where the affected individual (UW57-3) carries compound heterozygous TMEM67 mutations and a missense NINL mutation. The phenotype of the affected individuals is detailed in italic on each pedigree under the corresponding mutations. MTS Molar Tooth Sign, DD Developmental Delay, ESRF End-Stage Renal Failure.