Knocking out lca5 in zebrafish causes cone-rod dystrophy due to impaired outer segment protein trafficking.

Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1-2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7 dpf lca5-/- zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5-/- zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish.

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.

Knockout of ush2a gene in zebrafish causes hearing impairment and late onset rod-cone dystrophy.

Most cases of Usher syndrome type II (USH2) are due to mutations in the USH2A gene. There are no effective treatments or ideal animal models for this disease, and the pathological mechanisms of USH2 caused by USH2A mutations are still unknown. Here, we constructed a ush2a knockout (ush2a-/-) zebrafish model using TALEN technology to investigate the molecular pathology of USH2. An early onset auditory disorder and abnormal morphology of inner ear stereocilia were identified in the ush2a-/- zebrafish. Consequently, the disruption of Ush2a in zebrafish led to a hearing impairment, like that in mammals. Electroretinography (ERG) test indicated that deletion of Ush2a affected visual function at an early stage, and histological analysis revealed that the photoreceptors progressively degenerated. Rod degeneration occurred prior to cone degeneration in ush2a-/- zebrafish, which is consistent with the classical description of the progression of retinitis pigmentosa (RP). Destruction of the outer segments (OSs) of rods led to the down-regulation of phototransduction cascade proteins at late stage. The expression of Ush1b and Ush1c was up-regulated when Ush2a was null. We also found that disruption of fibronectin assembly at the retinal basement membrane weakened cell adhesion in ush2a-/- mutants. In summary, for the first time, we generated a ush2a knockout zebrafish line with auditory disorder and retinal degeneration which mimicked the symptoms of patients, and revealed that disruption of fibronectin assembly may be one of the factors underlying RP. This model may help us to better understand the pathogenic mechanism and find treatment for USH2 in the future.