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

Abstract

M 2019, 'Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish', AbstractMutations 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 nonphotoreceptor 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.

Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

“…[ 29 ] As shown by our results, rod specific CRX and RHO were down‐regulated, whereas cone‐specific PDE6c and GNAT2 showed a four‐fold increase in the edited mouse retina (Figure 6g, red bars). Expression of OPN1MW was not altered when compared to control retina, previous corroborating data that disruption of NR2E3 in the adult mouse retina would not affect some previously known rod specific genes regulated by NR2E3 , [ 29,31 ] which might be caused by the epigenetic modification in the adult mouse retina. [ 29 ] Here we showed that the optimized dual‐AAV split‐ABE could effectively generate precise A‐to‐G editing and achieve gene abruption through AI‐MAST strategy to modulate gene expression in non‐proliferating retinal cells.…”

Nr2e3 functional domain ablation by CRISPR-Cas9D10A identifies a new isoform and generates Retinitis Pigmentosa and Enhanced S-cone Syndrome models

“…The differences in the degenerative process are also significant when analyzing the markers of retinal stress, GFAP and Iba1 (Figure 4). However, we did not observed differences in the caspase-3 marker between the wild type and mutant retinas indicating degeneration is not caused by apoptosis (data not shown), in accordance with studies in the rd7 mouse model and the knockout in zebrafish, where TUNEL assays were not significative (Cheng et al, 2011;Xie et al, 2019). Aberrant photoreceptor structure and disk packaging ( Figure 3B In contrast, the D27 mutation clearly produces two proteins, the long isoform lacking the dimerization domain, and the short isoform at much higher levels than in the WT retina.…”

“…However, some authors question rd7 as a complete model of the human disease due to some discordances between mouse and human functional tests ). Knockout lines of Nr2e3 have been generated in mice (Webber et al, 2008) and zebrafish (Xie et al, 2019) resembling some of the phenotypic features found in the rd7 retina. Even so, the molecular mechanisms causing the diverse phenotypes of Nr2e3-associated pathologies are still unknown.…”

Novel Pathogenic Sequence Variants in NR2E3 and Clinical Findings in Three Patients

“…The loss of Nr2e3 also leads to the de-repression of cone specific genes in rods and Chen et al suggest that the misexpression of many of these cone transcripts may have toxic consequences for the rod cells and can result in the degeneration of the rods that is seen in patients with NR2E3 variants. However, Xie et al did not identify an excess number of S-cones using Nr2e3 knock out zebrafish, created using CRISPR technology [37]. They demonstrated both the normal development and number of cones, with no excess of S-cones, and showed that the L and M cones progressively degenerated.…”

“…They demonstrated both the normal development and number of cones, with no excess of S-cones, and showed that the L and M cones progressively degenerated. They also demonstrated that rod precursors failed to differentiate into rods; rod-specific genes were not expressed, and rod outer segments did not develop [37].…”