The nuclear receptor transcription factor Nr e is expressed
The nuclear receptor transcription factor Nr2e3 is expressed in photoreceptors. It forms a complex with Crx that enhances the Selamectin of rod-specific genes and represses the expression of cone-specific genes in rods [25,26]. Mutations in human Nr2e3 cause ESCS, an autosomal recessive disorder characterized by reduced rod, L- and M-cone responses and enhanced sensitivity to short-wave light due to supernumerary S-cones. In addition, patients with ESCS have dysmorphic retinas, showing an early disorganization of the laminated retinal structure with rosette formation in the outer nuclear layer (ONL) where photoreceptor cell bodies are located, and a slow, progressive retinal degeneration [, , , ]. Mutations in Nr2e3 have also been identified in patients with Goldmann-Favre syndrome, clumped pigmentary retinal degeneration , adRP , and arRP . A spontaneous recessive null mutation in the murine Nr2e3 has been identified in the retinal degeneration 7 (rd7) mice. The phenotype of this mutant includes progressive photoreceptor degeneration and attenuation of the electroretinogram (ERG) with time, abnormal retinal lamination with rosette formation in the ONL and a 2-fold increase in the number of S-cones [34,35]; however, the majority of the photoreceptors in the rd7 retina are a hybrid cell type that expresses both rod- and cone-specific genes .
The roles played by Nrl and Nr2e3 in rod/cone cell fate determination and differentiation have been exploited in a strategy of therapeutic cellular reprogramming to treat RP, a retinal degeneration characterized by primary rod defects and degeneration followed by secondary cone loss. Many RP-causing mutations occur in rod-specific genes. The rationale for such a strategy is that knocking down rod determinants such as Nrl or Nr2e3 reprograms rods to a cone fate, rendering rod-specific gene mutations irrelevant, with consequent preservation of retinal structural integrity and function [, , , ].
Although much is known about the function of Nr2e3, major questions remain unanswered. For example, the role of Nr2e3 in retinal progenitors for cell fate determination is unclear . In Nr2e3 retinas, the aberrant S-cones could have arisen from an alteration of cell fate or dedifferentiation of postmitotic premature rods .
Given the strategic importance of these transcription factors in potential therapeutic intervention, it is important to better understand their function. We have decided to study Nr2e3 in zebrafish by knocking out the gene using clustered regularly interspaced short palindromic repeat (CRISPR) technology. Architecture and cell types in zebrafish retina are conserved with that in human; furthermore, the zebrafish is diurnal and possesses a cone dominant retina similar to the macula in human retina [, , ]. Importantly, genomic structure and amino acid sequence of zebrafish Nr2e3, especially the DNA-binding domain (DBD) and ligand binding domain (LBD), is highly conserved with the human ortholog [25,44]. For these reasons, zebrafish could be a suitable organism to study the roles of Nr2e3 underlying photoreceptor development and differentiation. Here, we report on findings from the study of such Nr2e3-null zebrafish.
Materials and methods
Discussion Loss of Nr2e3 function leads to very different outcomes for rod development in mouse and in zebrafish. In the rd7 mouse, rod genes were expressed normally at the mRNA and protein levels [5,25,36,60]. The physiological function of rods was maintained initially . These observations are consistent with normal development of rods in the rd7 mouse. By contrast, the post-mitotic premature rods of Nr2e3 zebrafish fail to develop at all. The phenotype of Nr2e3 zebrafish resembles that of Nrl mouse in that deletion of the gene ablated expression of rod genes and prevented formation of rod OSs. The physiological function of rods was not detected . These data demonstrated that development of rods was prevented totally in Nrl mouse. Accordingly, analogous to Nrl's role in the murine retina, zebrafish Nr2e3 acts like a “molecular switch” in modulating rod-specific genes. The critical role Nrl and Nr2e3 each play in rod development in, respectively, mouse and zebrafish, may be reflected in the developmental order of expression of these two genes. In mouse, Nrl transcripts appear at ~E12.5  and before Nr2e3 at ~E16.5 , while in zebrafish the expression of Nr2e3 is earlier than Nrl in rod lineage. Nr2e3 appears first in rod progenitors in the INL and in all rod precursors in the ONL. Nrl transcripts appear only in the ONL . Unlike the situation in mouse where Nrl can activate expression of Nr2e3 , the earlier appearance of Nr2e3 transcripts in zebrafish means that its expression is not activated by Nrl. ESCS patients present with severe deficit of rod function at an early stage and histological analysis of one postmortem retina revealed an absence of rods [26,27,57]. Furthermore, retinal structural analysis led to the conclusion that rods failed to differentiate in ESCS retinas . During fetal development, Nr2e3 expression follows Nrl very closely, and mutations in either Nrl or Nr2e3 can lead to ESCS , suggesting these two genes may play parallel roles in rod development.