Fig. 3

Morphological, functional and epistatic effects of ninl and dzank1 knockdown in zebrafish retina.

(a-c) Images of 4 dpf living zebrafish. Un-injected controls (WT) appear morphologically normal (a), while embryos injected with 2 ng of ninl atgMO display morphological defects, including ventrally curved body axis and small eyes (b). Embryos injected with 6 ng dzank1 ex8 spMO resulted in expanded melanophores, small eyes and severe pericardial edema (c) (a'-c') Retinal histology of 4 dpf zebrafish morphants examined by cryo-sections, where bodipy highlights the OS (red) and nuclei are stained with DAPI (blue) in all panels. Outer segments were shortened and dysmorphic in ninl and dzank1 morphants compared to wildtype larvae. (d-g) Ninl interacts genetically with dzank1. Injection of sub-effective dzank1 (1 ng/nl) MO (e') or ninl (0.5 ng/nl) MO (f') shows normal OS shape and length in morphologically normal appearing larvae, which could not be distinguished from un-injected embryos (WT) or control MO-injected larvae (d'). Combined injection of sub-effective concentrations of ninl (0.5 ng/nl) and dzank1 (1 ng/nl) MO together results in almost complete absence of OS (g'). (h) Quantification of Outer Segment length, shown as a scatter graph where each datapoint represents the mean OS length in one larva, revealed a significantly decreased length of outer segments in ninl (2ng/nl), dzank1 (6ng/nl) and ninl/dzank1 double morphants as compared to controls. Bars represent the mean value for each treatment group with the Standard error of the mean (SEM) ***P<0.0001, ** P<0.001, unpaired Student's t-test. (i) Analysis of the Opto Kinetic Response (OKR) showing severely decreased responses in larvae injected with 2 ng ninl atgMO or 6 ng dzank1 ex8 spMO (*** p<0.0001, Student's t-test). Scale bars represent 500 μm (a-c and e-h) and 15 μm (a'-c' and e'-h'). RPE, retinal pigment epithelium; OS, Outer Segment; IS, Inner Segment; ONL, Outer Nuclear Layer; OPL, Outer Plexiform Layer.