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Rod genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor composition and progressive retinal degeneration

['Fei Liu', 'Key Laboratory Of Molecular Biophysics Of Ministry Of Education', 'College Of Life Science', 'Technology', 'Huazhong University Of Science', 'Wuhan', 'P.R. China', 'State Key Laboratory Of Freshwater Ecology', 'Biotechnology', 'Institute Of Hydrobiology']

Date: 2022-05

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.

Vision is mediated by two types of light-sensing cells named rod and cone photoreceptors in animal eyes. Abnormal generation, dysfunction or death of photoreceptor cells all cause irreversible vision problems. NRL is an essential gene for the generation and function of rod cells in mice and humans. Surprisingly, we found that in the zebrafish, a popular animal model for human diseases and therapeutic testing, there are two types of rod cells, and eliminating the function of nrl gene affects the rod cell formation at the embryonic stage but not at the juvenile and adult stages. The rod cell formation at the post-embryonic is driven by the mafba gene, which has not been reported to play a role in rod cells. In addition to the reduced number of rod cells, deletion of nrl also results in the emergence of rod/green-cone hybrid cells and an increased number of green cones. The ensuing cellular and molecular alterations collectively lead to retinal degeneration. These findings expand our understanding of photoreceptor development and maintenance and highlight the underlying conserved and species-specific regulatory mechanisms.

Funding: This work was supported by the National Key Research and Development Program of China [2018YFA0801000] and the National Natural Science Foundation of China [31601026, 81670890, 31871260 and 31801041]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we generated an nrl knockout zebrafish model by using the CRISPR-Cas9 technology and systematically investigated the developmental processes of rod photoreceptors. Surprisingly, we found two waves of rod genesis in zebrafish, which could be distinguished as nrl-dependent at embryonic stage and nrl-independent at post-embryonic stage. In addition, we observed a continuous increase of green cones in adult nrl knockout zebrafish. The cellular and molecular mechanisms underlying this unexpected retinal phenotype were further investigated, and a modified model for the developmental processes and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish was established.

In recent years, the zebrafish has emerged as a useful animal model for retinal degeneration and regeneration studies [ 24 – 27 ]. Interestingly, increasing evidence suggests that the current working model of photoreceptor development, which is largely based on studies in mice and humans, does not perfectly match the observations in the zebrafish [ 28 – 31 ]. For example, a portion of rods are derived from S-cone precursors in mice, but this phenomenon is not observed in the zebrafish [ 29 ]. In addition, our recent study has shown that deletion of nr2e3, another key factor determining rod fate [ 32 , 33 ], eliminates rods completely but does not increase the number of UV or blue cones (corresponding to S-cones) in the zebrafish [ 30 ]. This observation disagrees with the retinal phenotype of Nr2e3 or Nrl knockout mice [ 10 , 32 ]. Whether rods can be derived from the precursors of S cones or other types of cones in zebrafish, and if so, how this process is regulated remain unclear.

Neural retina leucine zipper (NRL) is a large MAF family transcription factor expressed in the retina and pineal gland [ 8 , 9 ], and has been identified as the master gene determining the cell fate between rod versus cone photoreceptors in mammals. Disruption of Nrl in mice results in the complete loss of rod photoreceptors, accompanied by the presence of a large excess of S-cone-like photoreceptors [ 10 , 11 ]. Similarly, loss-of-function mutations in NRL cause the autosomal recessive enhanced S-cone syndrome (ESCS), which is characterized by the absence of rod response and enhanced S-cone function, in humans [ 12 – 14 ]. Moreover, ectopic expression of Nrl transforms the fate of cone precursors to rods in mice [ 15 ], suggesting that NRL is essential and sufficient for the fate determination and differentiation of rod photoreceptors. Additionally, NRL activates the expression of many rod-specific genes (such as RHO, GNAT1, PDE6A, REEP6, and MEF2C) synergistically with CRX and NR2E3 [ 16 – 21 ], and represses the cone-specific genes (such as Thrb and Opn1sw in mice) either directly or through activating the expression of NR2E3 [ 22 , 23 ].

Vision is involved in many fundamental behaviors of animals such as navigation, foraging, predator avoidance, and mate choice [ 1 ]. In humans, inherited retinal diseases are a major cause of irreversible vision impairment and blindness, causing serious physical discomfort and mental problems in the patients, who also face a heavy economic burden [ 2 , 3 ]. There are two types of light-sensing cells named rod and cone photoreceptors in vertebrate eyes. Rods are highly sensitive to dim light and function under conditions of low light, whereas cones respond to bright light and mediate color vision [ 4 , 5 ]. Rods and cones are derived from the same progenitor cells, which are regulated by several extrinsic signals and intrinsic transcription factors, such as CRX, NRL, and NR2E3 [ 6 , 7 ]. Exploring the fundamental mechanisms controlling the fate determination and differentiation of photoreceptors in diverse animal models is of great value for understanding the development and evolution of photoreceptors and the pathogenesis of retinal degenerative diseases.

Progressive photoreceptor degeneration has been reported to activate regeneration in zebrafish [ 50 ]. Thus, we speculated that in the nrl-KO zebrafish, retinal regeneration might also be activated to replenish the lost rods. Immunostaining for Pcna on retinal sections indeed showed more proliferating cells in the nrl-KO retinas than in the WT controls since 9 mpf ( Fig 10E and 10F ). These results suggested that the progressive retinal degeneration in the nrl-KO zebrafish induces regeneration of photoreceptors in an nrl-independent manner.

(A) The TUNEL assay results revealed that multiple types of retinal cells, including rods, cones, RPE, and inner retinal cells, undergo apoptosis in the nrl-KO retinas. Representative images are shown. Scale bar: 20 μm. (B) Quantitation of the apoptotic cells per section from 5 mpf to 13 mpf. The results are shown as mean with SD (n = 3). *, p < 0.05; **, p < 0.01. (C) RPE morphology in the WT and nrl-KO zebrafish at 20 mpf are shown, as assessed by immunostaining the retinal whole-mounts for ZO-1 Scale bar: 20 μm. (D) The up-regulation of GFAP in nrl-KO retinas as detected by immunostaining. CN, cone nuclear layer; RN, rod nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 25 μm. (E) Regeneration of photoreceptors was more active in the nrl-KO retinas at 9 mpf and 18 mpf, as reflected by the increase in the number of Pcna-positive cells (proliferating cells) in the ONL, compared with the WT levels. Scale bars: 50 μm. (F) Quantitation of the Pcna + cells located in the ONL per section. The results are shown as mean with SD (n = 4) from 7 dpf to 29 mpf. *, p < 0.05; **, p < 0.01.

The gradual loss of rods is considered a main cause of the secondary death of cones in retinitis pigmentosa patients [ 48 , 49 ]. As the nrl-KO zebrafish showed a significantly reduced number of rods and over-growth of green cones, we assessed whether these developmental abnormalities lead to retinal degeneration. TUNEL assay was performed to evaluate cell death on retinal sections from 5 mpf to 13 mpf. Indeed, we observed more apoptotic cells in the nrl-KO retinas, including rods (predominantly), cones, RPE cells, and inner retinal cells ( Fig 10A and 10B ). Immunostaining for ZO-1 showed the abnormal morphology of some RPE cells in the nrl-KO retinas ( Fig 10C ). We also detected the up-regulation of GFAP, a hallmark of retinal damage, in aged nrl-KO retinas ( Fig 10D ). These results demonstrated the nrl-KO zebrafish undergo a slowly progressing retinal degeneration.

The genes nearby mafba and their relative orders were extracted from the genomes of a wide range of vertebrates, including ancient fishes (sea lamprey, elephant shark, spotted gar, and coelacanth), modern fishes (European eel, zebrafish, Japanese medaka, and torafugu), amphibians (tropical clawed frog), reptilians (American alligator), birds (chicken), and mammals (house mouse and human). The mafba and mafbb genes appear to have originated from the ancestral gene mafb through genome duplication specifically in teleost fishes.

To assess whether the function of mafba in rod development also exists in other species, the evolutionary origin of mafba was investigated via gene collinearity analysis. We found that the mafba and mafbb genes presumably arose through the duplication of an ancestral mafb gene specifically in the teleost fish ancestor ( Fig 9 ). This genetic feature is not seen in ancient fishes, amphibians, reptilians, birds, or mammals. From European eel to zebrafish, there seems to be a chromosomal rearrangement upstream of the mafbb gene. The biological roles of the two genes may have gradually diverged during evolution, whereby mafba may have acquired the unique role in the rod development in modern fishes such as zebrafish and medaka.

To validate whether mafba plays a role in rod development, we knocked out this gene in zebrafish by using the CRISPR-Cas9 technology ( S8B and S8C Fig ), and then generated an nrl and mafba double-knockout line by crossing the two single-knockout lines. The homozygous mafba mutants died at 9–10 dpf with multi-organ developmental defects, as described previously [ 46 , 47 ]. In the nrl-KO zebrafish, deleting both copies of mafba completely eliminated rods, based on the results of fluorescence analysis at 9 dpf ( Fig 8B ). Interestingly, deleting one copy of mafba also dramatically retarded the development of nrl-independent rods at 9 and 15 dpf, suggesting a dose-dependent effect of mafba ( Fig 8B ). We also measured the mRNA levels of nrl, nr2e3, and mafba through RNA-seq and qPCR. The expression of mafba in the nrl-KO retinas was significantly decreased at 2 mpf and 5 mpf ( Fig 8C ), excluding the genetic compensation effect caused by nrl disruption. Taken together, our results demonstrated that mafba is a novel regulatory gene driving rod genesis in the zebrafish.

(A) Clustering analysis of the large MAF genes and the genes involved in photoreceptor development by using the expression data from scRNA-seq. The cell-type-specific expression pattern of mafba was highly similar to those of nrl and nr2e3. (B) The distribution of rods (labeled with EGFP) in the nrl-KO zebrafish carrying the WT, heterozygous, and homozygous mafba alleles at 9 dpf (left panel) and 15 dpf (right panel). Knocking out mafba further reduced the genesis of rods in the nrl-KO zebrafish. The dotted circles indicate the boundaries of the retinas. Scale bars: 100 μm. (C) The expression levels of nrl, nr2e3, and mafba in the nrl-KO retinas at 2 mpf and 5 mpf were measured via RNA-seq and qPCR. The data are shown as mean with SD (n = 3). *, p < 0.05; **, p < 0.01.

A reasonable explanation for the occurrence of nrl-independent rods is that other genes might at least partly substitute for the functions of nrl in driving rod genesis. Genes belonging to the large MAF family (MAFA, MAFB, and MAF/c-MAF) are the most likely candidates due to their high homologies with NRL [ 29 , 44 ]. In addition, MAFA has been reported to induce the expression of rod-specific genes when ectopically expressed in mice [ 29 , 45 ]. By analyzing our scRNA-seq data, the expression patterns of all large MAF genes and genes involved in photoreceptor development were determined. We found that mafba, one of the two MAFB orthologs in zebrafish, showed a closer cell-type-specific expression pattern to that of nrl and nr2e3 than the other MAF candidates (see the first three rows in Fig 8A ). Furthermore, the mafba gene (indicated by green bars in S8A Fig ) was the only large MAF family gene expressed in all the three types of rods at levels comparable to those of nrl and nr2e3.

Previous studies have shown the abnormal chromatin morphology of the S-cone-like cells in Nrl or Nr2e3 knockout mice [ 10 , 32 ]. Interestingly, our scRNA-seq data showed that the hmgn2 gene, which encodes a non-histone nucleosome binding protein and contributes to chromatin plasticity and epigenetic regulation [ 43 ], was remarkably up-regulated in nrl-KO rods. Through in situ hybridization, we observed that hmgn2 was expressed at a very low level in WT rods ( S6 Fig ). However, in the nrl-KO zebrafish, the expression of hmgn2 was specifically enhanced in the rods ( S7 Fig ). These results suggested that epigenetic modification is also involved in the formation of the intermediate photoreceptors in nrl-KO zebrafish.

To directly visualize these intermediate photoreceptors, immunostaining was performed on retinal sections from the Tg(rho:EGFP) transgenic zebrafish at 3 mpf with the anti-Opn1mw (green-cone opsin) antibody. Mis-expression and accumulation of Opn1mw in some rods (labeled by EGFP) were observed only in the nrl-KO retinas ( Fig 7E ). These results demonstrated the existence of rod/green-cone intermediate photoreceptors in the nrl-KO zebrafish.

The gene expression signatures of the three rod subtypes were characterized based on the gene expression profiles through unsupervised clustering analysis. The top 40 discriminative genes between the WT and nrl-KO rods are shown in a clustering heatmap ( Fig 7D ). The cells with high expression of rod-specific genes (such as rho, rhol, saga, sagb, and gnat1) and very-low expression of cone-specific genes (such as opn1mw1, opn1mw3, arr3a, grk7a, and gucy2d) represent the “typical” rods (leftmost in Fig 7D ) in WT zebrafish. By contrast, the cells with relatively low expression of rod-specific genes and high expression of cone-specific genes (especially the green-cone opsins) most likely represent the “hybrid” rods (rightmost in Fig 7D ) in the nrl-KO zebrafish.

The percentages of all the cell types in WT and nrl-KO retinas were analyzed ( Fig 7B ). In nrl-KO zebrafish, the percentage of rods was reduced by approximately 50%, whereas cones (more accurately the red/green cones) showed an obvious increase ( Fig 7B and 7C ). Three rod subtypes (rod-1, rod-5, and rod-11) were identified in our scRNA-seq analysis. The top 20 marker genes of these subtypes are shown in S1 Table . The rod-1 population (accounting for 80% of all the rods) was the major subtype of rods in WT zebrafish, but the percentage was reduced to 13% in the nrl-KO zebrafish. Conversely, the percentages of rod-5 and rod-11 populations were increased to 34% and 16% of all the rods in the nrl-KO zebrafish, respectively. These data suggested that knockout of nrl affects both the fate determination between rods versus green cones and the ratios of rod subtypes.

(A) tSNE visualization of the unsupervised cell clusters from 5-month-old WT and nrl-KO zebrafish. Left, the distribution of cell clusters between the WT and nrl-KO groups. Right, the retinal cell types identified via scRNA-seq (see also S6 Fig ). BC, bipolar cells; Cone_R/G, red and green cones; Cone_B/UV, UV and blue cones; RGC, retinal ganglion cells; HC, horizontal cells; AC, amacrine cells; RPE, retinal pigment epithelium. (B) Proportions of each retinal cell type in the WT and nrl-KO zebrafish. (C) The cell compositions and proportions of photoreceptor sub-clusters in WT and nrl-KO retinas. (D) The heatmap shows the clustering pattern of the top 20 down-regulated and top 20 up-regulated genes in the WT and nrl-KO rods. Yellow, high expression; purple, low expression. (E) Mis-expression of green-cone opsin in a proportion of rods in the nrl-KO retinas. The dorsal retinal regions of WT and nrl-KO zebrafish at 3 mpf are shown. Green, EGFP-labeled rods. Red, immunofluorescence signals of the anti-Opn1mw antibody. The green fluorescent signal in the nrl-KO group was artificially enhanced to discern the morphology of rods. ROS, outer segments of rods; GCOS, outer segments of green cones; RN, rod nuclear layer. Scale bar: 20 μm.

The retinal cell compositions and cell-type-specific gene expression patterns in WT and nrl-KO zebrafish at 5 mpf were characterized via single-cell RNA-seq (scRNA-seq) analysis. After filtering out invalid cells, 10374 cells (4682 from the WT group and 5692 from the nrl-KO group) were classified into 24 clusters via unsupervised cell clustering analysis ( Fig 7A ). The relative mRNA levels of all the genes expressed in the 24 cell types were determined ( S3 Dataset ). According to the expression patterns of known marker genes of each retinal cell type [ 39 – 42 ], the 24 cell clusters were identified as rods (3 subtypes), cones (2 subtypes), horizontal cells, bipolar cells (12 subtypes), amacrine cells, retinal ganglion cells (RGCs), retinal pigment epithelium (RPE) cells, Müller glia, astrocytes, and microglia ( S5 Fig and S4 Dataset ).

To validate whether the number of green-cones is increased, we performed immunostaining using the anti-Opn1mw (zebrafish green-cone opsin) antibody on retinal sections from WT and nrl-KO zebrafish. No obvious changes were observed in the nrl-KO retinas at 2 mpf and 3 mpf, except for some newly formed green-cone outer segments located below the normal single-row arrangement ( Fig 6D ). However, at 13 mpf, 2–3 rows of green-cone outer segments could be observed ( Fig 6D ), suggesting a progressive increase in the number of green-cones. The expression levels of genes involved in cone development (such as rx1, tbx2a, tbx2b, six7, and thrb) were not significantly affected as suggested by the RNA-seq data.

(A) The protein levels of rod-specific (rho, gnat1, and gnb1) and cone-specific (gnat2 and gnb3) genes in WT and nrl-KO retinas from 14 dpf to 3 mpf were evaluated using western blotting. Tubulin was used as a loading control. The asterisk indicates a non-specific band. (B) Quantitative analysis of the protein levels of rod- and cone-specific genes based on at least three independent experiments. WT samples, blue color; nrl-KO samples, red color. Data from different ages are arranged from left to right (from 14 dpf to 3 mpf) and indicated by different fill patterns. The data are shown as mean with SD (n = 3). *, p < 0.05. **, p < 0.01. (C) The mRNA levels of the rod and cone opsins in the WT and nrl-KO retinas at 2 mpf and 5 mpf were measured using qPCR. The data are shown as mean with SD (n = 3). ns, non-significant; *, p < 0.05; **, p < 0.01. (D) Detection of green-cones on retinal sections of WT and nrl-KO zebrafish at 2 mpf, 3 mpf, and 13 mpf by immunostaining using the anti-Opn1mw antibody. The dorsal retinal regions are shown. White arrows indicate the mislocalized green-cone outer segments. GCOS, green-cone outer segments; CN, cone nuclear layer; RN, rod nuclear layer; INL, inner nuclear layer. Scale bars: 50 μm.

To confirm the RNA-seq results, we measured the protein levels of representative rod-specific (rho, gnat1, and gnb1) and cone-specific (gnat2 and gnb3) genes from 14 dpf to 3 mpf via western blotting. The rod-specific genes were significantly down-regulated in the nrl-KO retinas ( Fig 6A and 6B ). In contrast, protein levels of the cone-specific genes were unchanged before 2 mpf, and significantly increased at 3 mpf ( Fig 6A and 6B ). We also performed qPCR to measure the mRNA levels of cone opsin genes at 2 mpf and 5 mpf. The green-cone opsins (opn1mw1, opn1mw2, opn1mw3, and opn1mw4) were significantly up-regulated at both time points, whereas the expression of UV-, blue-, and red-cone opsins were almost unchanged ( Fig 6C ). These results correlated well with our RNA-seq data and suggested that the down-regulation of rod-specific genes and up-regulation of cone-specific genes (more particularly green-cone genes) both occur in the nrl-KO zebrafish.

To our surprise, only few differentially expressed genes (aipl1, nxnl1, sagb, gucy2f, rpgra, and cnga3b) were closely related to the development or degeneration of retinas. Because we observed a reduced number of rods in the nrl-KO zebrafish, we directly examined the expression of rod- and cone-specific phototransduction genes by using our RNA-seq data. As expected, most of the rod-specific genes were down-regulated (adjusted p-value ≤0.05) in the nrl-KO group ( Fig 5B ). However, the fold changes were mostly less than 2, which were ignored by the routine bioinformatic pipelines. Meanwhile, the mRNA levels of cone-specific genes were not significantly changed, except for the obviously up-regulated green-cone opsins ( Fig 5C ).

(A) The volcano plot shows the 386 differentially expressed genes (196 up-regulated and 190 down-regulated) between WT and nrl-KO retinas at 2 mpf identified via RNA-seq. The red and blue points indicate the up-regulated and down-regulated genes, respectively. (B) The functional categories enriched among the top 100 differentially expressed genes. (C) The expression patterns of rod- and cone-specific phototransduction genes in nrl-KO retinas shown as a heatmap. Most of the rod genes were down-regulated in the nrl-KO retinas.

To investigate the transcriptome dynamics in the nrl-KO retinas, we performed RNA-sequencing (RNA-seq) analysis on 2 mpf WT and nrl-KO retinas. A total of 386 differentially expressed genes (fold change ≥2, adjusted p-value ≤0.05, and FPKM ≥1) were detected ( Fig 5A and S1 Dataset ). The functions and related biological processes of the top 100 differentially expressed genes were classified into several categories ( S2 Dataset ). Interestingly, we found that the most affected functional categories were inflammatory and immune response, signal transduction, metabolic enzymes, cell adhesion and extracellular matrix, transport, and cell protection and death ( Fig 5B ). These altered biological functions were similar to those identified in the Nrl knockout mice [ 38 ].

To discern the EGFP-labeled rods more clearly, retinal sections were prepared from the Tg(rho:EGFP) transgenic zebrafish at 1 mpf and observed at high magnification ( Fig 4B ). The nrl-KO groups had fewer rods than the WT controls, as suggested by the thinner rod nuclear layer ( Fig 4B ). Besides, the regions nearby ciliary marginal zone (labeled with boxes in Fig 4B ) in the nrl-KO retinas showed no or little fluorescence signal of rods, consistent with our immunostaining results shown above ( S2B Fig ). Taken together, these results suggested that there are two waves of rod genesis in zebrafish. Knockout of nrl eliminates the first wave of rod genesis, starting at about 3 dpf (embryonic stage), but does not affect the second wave of rod genesis, starting at 7–10 dpf (juvenile stage). Accordingly, we named them as the nrl-dependent and nrl-independent rods, respectively.

The protein levels of Rho (rhodopsin) were detected by dot blotting in WT and nrl-KO zebrafish ( S4 Fig ). Rho expression was indeed reduced in the nrl-KO retinas. Transmission electron microscopy was performed to determine whether the ultrastructure of the rod outer segments was affected in the nrl-KO zebrafish. There was no large difference in the length of the outer segments between nrl-KO and WT rods at 9 mpf ( S5 Fig ).

(A) The Tg(rho:EGFP) transgenic line was used to label rods with EGFP. Representative images of flattened whole-mount retinas from WT and nrl-KO zebrafish at 1 mpf, 2 mpf, and 3 mpf are shown. The dashed lines indicate the edges of the retinas. Scale bars: 200 μm. (B) EGFP-labeled rods were observed on the retinal sections from WT and nrl-KO Tg(rho:EGFP) transgenic zebrafish at 1 mpf. The overall views are shown in the upper panel. The regions nearby the ciliary marginal zone (labeled with boxes) showed no or little fluorescence signal of rods. Enlarged images of the dorsal retinal regions are shown in the lower panel. ROS, rod outer segment; CN, cone nuclear layer; RN, rod nuclear layer; INL, inner nuclear layer; GCL: ganglion cell layer. Scale bars: 50 μm.

Rods were labeled with EGFP by crossing the WT or nrl-KO zebrafish with the Tg(rho:EGFP) transgenic line. Fluorescence was observed every 2 days from 3 dpf. Representative images of WT and nrl-KO retinas are shown in (A) for 3–9 dpf and (B) for 11–17 dpf. The dotted circles indicate the boundaries of the retinas. D, dorsal; V, ventral; N, nasal; T, temporal. Scale bars: 100 μm. (C) Immunostaining of retinal sections for Gnat1 (the rod transducin alpha-subunit) showed exact co-localization with EGFP in rods at 7 dpf. Rods were barely observed in the nrl-KO retinas. Scale bar: 50 μm.

To trace the developmental processes of rods in the nrl-KO zebrafish, we used the Tg(rho:EGFP) transgenic zebrafish to label rods with EGFP (enhanced green fluorescent protein) in vivo. In WT zebrafish, rods first appeared in the retinal ventral edge and then spread to the entire retina with a rapidly increasing fluorescence intensity ( Fig 3A and 3B ). However, the fluorescence of rods in the nrl-KO zebrafish was undetectable or close to the background level until 7 dpf ( Fig 3A ). Interestingly, after 10 dpf, the fluorescence of rods in the nrl-KO zebrafish became obvious and rapidly increased with age ( Fig 3B ). Immunostaining the retinal sections for another rod-specific protein Gnat1 also showed the nearly complete absence of rods in the nrl-KO retinas at 7 dpf ( Fig 3C ). Flattened retinal whole-mounts of WT and nrl-KO zebrafish from 1 mpf to 3 mpf showed an increasingly extensive distribution of rods with age ( Fig 4A ), although the rod opsin expression level (reflected by the fluorescence intensity) was still lower in the nrl-KO retinas at 3 mpf than in age-matched WT controls ( Fig 4A ).

(A) The rod outer segments were visualized via immunostaining on the flattened whole-mount retinas from WT and nrl-KO zebrafish at 2 mpf using the anti-Rho antibody. The overall views of WT and nrl-KO retinas are shown in the low-magnification images. Scale bar: 200 μm. (B) High-magnification images from the dorsal and ventral retinal regions are shown. The density of rods is reduced in the nrl knockout retinas, especially in the ventral retinal region. Scale bar: 50 μm.

Deletion of Nrl has been reported to eradicate rods in mice [ 10 , 11 ]. To assess whether the nrl-KO zebrafish has rods, immunofluorescence analysis with the anti-zebrafish Rho antibody was performed on retinal sections from WT and nrl-KO zebrafish. Indeed, we could detect the rod outer segments from 14 dpf (day post-fertilization) until 18 mpf (month post-fertilization) (Figs 1D and S2 ). The rod outer-segment layer was significantly thinner in the nrl-KO zebrafish than in the WT zebrafish ( S3A Fig ). Additionally, retinal whole-mount immunostaining revealed a diluted pattern of rod outer segments in the nrl-KO zebrafish, especially in the ventral retina (Figs 2 and S3B ). Interestingly, we noticed that the rod generation in the marginal regions of nrl-KO retinas was delayed at 14 dpf and 1 mpf ( S2A and S2B Fig ). However, at 2 mpf and 4 mpf, these regions were covered by rods as in other retinal regions ( S2C and S2D Fig ), suggesting a central-to-peripheral rod development pattern in the nrl-KO zebrafish. Our results indicated that knockout of nrl in zebrafish reduced the number of rods rather than completely eliminating them, which was quite different from the retinal phenotypes of Nrl and Nr2e3 knockout mice [ 10 , 37 ].

Histological analysis was performed to examine the retinal morphology of the nrl-KO zebrafish. Surprisingly, the presence of rod nuclei (labeled as RN in S1B and S1E Fig ) suggested that rods are not completely eliminated by nrl knockout in zebrafish. We also observed that the photoreceptor and outer-segment layers were thinned in the nrl-KO retinas ( S1 Fig ). The rosette-like anomalies commonly observed in the outer nuclear layer (ONL) of Nrl or Nr2e3 knockout mice [ 35 , 36 ] were not observed in the nrl-KO zebrafish ( S1A and S1D Fig ).

(A) The gene structure of zebrafish nrl and the CRISPR-Cas9 target site used for knocking out nrl are shown. Orange boxes, the exons encoding the MTD and bZIP domains; red arrow, the CRISPR-Cas9 target site; black arrows, the primers used for mutation detection. (B) Sequencing validation of the homozygous nrl del8 mutation (c.230_237del8). (C) The nrl mRNA levels in the nrl-KO zebrafish were measured using qPCR. The data are shown as mean with SD (n = 3). *, p < 0.05. (D) Immunostaining of retinal sections from WT and nrl-KO zebrafish from 1 mpf to 18 mpf with the anti-Rho antibody for the rod outer segments. Scale bars: 25 μm. ROS, rod outer segment; CN, cone nuclear layer; RN, rod nuclear layer; INL, inner nuclear layer; GCL: ganglion cell layer.

The nrl knockout zebrafish was generated via CRISPR-Cas9 targeting the exon 2 of nrl, which encodes the conserved N-terminal minimal transactivation domain ( Fig 1A ). Through several rounds of crossing and mutation screening, we obtained a homozygous nrl mutant zebrafish line carrying a frameshift mutation (c.230_237del8, p.P77Hfs*4) ( Fig 1B ). The mutation was predicted to result in a severely truncated Nrl protein without the DNA binding and transactivation domains. The nrl mRNA levels were significantly up-regulated in this mutant zebrafish line (referred to as nrl-KO hereinafter) ( Fig 1C ), possibly due to a negative feedback mechanism [ 34 ]. No alternative transcript of nrl was detected in the retinas of wild type and nrl-KO zebrafish. We also sought to detect the protein levels of Nrl in WT and nrl-KO retinas. Unfortunately, we could not obtain a working antibody against the zebrafish Nrl.

Discussion

The zebrafish has become a popular animal model for retinal degeneration and regeneration studies [24–26] and drug screening [51,52]. The developmental models regarding the photoreceptors in zebrafish are mainly based on the assumption that the functions of the key regulatory genes (such as NRL and NR2E3) are conserved between zebrafish and mammals. However, this assumption has been challenged by several recent studies [29–31]. In this study, we generated an nrl knockout zebrafish model and systematically investigated its retinal phenotype. We also characterized the retinal cell populations and the cell-type-specific gene expression profiles in both WT and nrl knockout zebrafish through single-cell RNA-seq. The unexpected retinal phenotype and the cellular and molecular findings described here expanded the current developmental model of rod photoreceptors in the zebrafish (Fig 11).

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TIFF original image Download: Fig 11. Working model for the development of rod photoreceptors in the zebrafish. There may be two types of rod precursors defined as nrl-dependent and nrl-independent in the zebrafish retinas. The former is responsible for the production of rods at embryonic stage, and knocking out nrl abolishes the differentiation into rods (left panel). The latter is responsible for the production of rods at the juvenile and adult stages. In the presence of nrl and mafba, these cells differentiate into rods. Knocking out nrl does not severely affect the production of rods from these cells but causes abnormal expression of rod- and cone-specific genes and a progressive increase of green cones (M-cones) with age. Knocking out both nrl and mafba eliminates all rods, suggesting that mafba also plays an important role in the fate determination of this type of rods (right panel). https://doi.org/10.1371/journal.pgen.1009841.g011

Interestingly, we found that some features of the nrl knockout zebrafish, such as the moderate downregulation of rod-specific genes and the presence of rod-cone “hybrid” cells, are in part similar to the phenotype of the rd7 mice carrying an Nr2e3 mutation [32]. Meanwhile, the nr2e3 knockout zebrafish has no rods and shows no expression of rod-specific genes [30], a phenotype similar to that of the Nrl knockout mice [10]. Moreover, Nr2e3 is a direct target gene of Nrl in humans and mice [23]. However, knockout of nrl does not significantly affect the expression of nr2e3 in zebrafish (Fig 8C). A previous study has shown that nr2e3 is expressed prior to nrl during the rod genesis in zebrafish [28]. Taken together, we speculate that nrl might act in parallel with or even downstream of nr2e3 in zebrafish. The functions of NRL and NR2E3 and the regulatory relationship between the two genes are not completely conserved between zebrafish and mammals.

The surprising retinal phenotype of the nrl-KO zebrafish indeed does not support the use of this zebrafish line as a suitable model of ESCS. However, the reduced number of rods and over-growth of green cones in the nrl-KO retinas could still cause a slowly progressing retinal degeneration, as validated by the elevated levels of apoptosis of retinal cells and significant up-regulation of GFAP in Müller glia (Fig 10). The gradual loss of rods results in secondary death of cones in retinitis pigmentosa, the most common type of inherited retinal degenerative disease [48,49]. In addition, our bulk and single-cell RNA-seq data may provide valuable clues for investigating the genes and pathways involved in the occurrence and progression of retinal degeneration. For example, several differentially expressed genes, including aipl1 (aryl hydrocarbon receptor interacting protein-like 1), ahrrb (aryl-hydrocarbon receptor repressor b), rpgra (retinitis pigmentosa GTPase regulator a), nxnl1 (nucleoredoxin like 1), have previously been associated with retinal degenerative diseases [53–57].

One of the major findings in this study is the discovery of two waves of rod genesis occurring at the embryonic and post-embryonic stages through nrl-dependent and -independent mechanisms, respectively. The second wave of nrl-independent rods may likely represent the rod population constantly generated from the Müller glia-derived unipotent progenitors during the growth of zebrafish [25,28,58], because they show similar developmental stages and retinal locations at the beginning of their genesis. Additionally, we found that the regeneration of rods from the injury-induced Müller glia-derived multipotent progenitors [25] may also be nrl-independent (Fig 8). In other words, nrl is likely not required for the generation of rods from Müller glial cells.

Interestingly, a recent study has reported the dispensable role of nrl in the specification of rods in adult zebrafish but not zebrafish larvae [59], which is partly consistent with our conclusions. Nevertheless, the two studies diverge in several aspects. Firstly, we demonstrated that even in the adult nrl knockout zebrafish, the number of rods is reduced to a certain extent, as validated through multiple types of cellular and molecular experiments. Secondly, the study of Oel et al has shown an increase of UV cones in the nrl mutants at 4 dpf. However, in this study, we found that the mRNA levels of UV opsin (opn1sw1) in the nrl-KO zebrafish were not up-regulated (Figs 5B and 6C). Lineage tracing experiments have shown that zebrafish rods are unlikely derived from UV- and blue-cone precursors [29]. Besides, our previous study has shown that knocking out nr2e3 in zebrafish does not increase the number of UV cones, too. Thirdly, we found that the number of green cones is increased in adult nrl knockout zebrafish in an age-related manner, and there are rod/green-cone hybrid cells in nrl-KO zebrafish. This phenomenon was not mentioned in the study of Oel et al [59]. Furthermore, we identified the mafba gene as a novel driving gene for the development of rods in zebrafish, which provided a reasonable explanation for the retinal phenotype of the nrl-KO zebrafish. Our study focused more on the temporal and spatial changes of retinas in the nrl knockout zebrafish at the tissue, cellular and molecular levels, as well as the regulatory mechanisms controlling these biological processes.

The gradual increase in the number of green cones and the presence of rod/green-cone “hybrid” photoreceptors suggest that there may be cell fate transformation between rods and green cones in the nrl knockout zebrafish. Among the four types of cone opsins, the green-cone opsin shows the highest similarity and the closest evolutionary relationship to the rod opsin in non-mammalian vertebrates [60]. Thus, we speculated that at least a proportion of rods and green cones may originate from the same precursors in zebrafish, and nrl may play a crucial role in the fate choice between rods versus green cones. However, the RH2 (green-cone opsin) and SWS2 opsins are thought to be lost in mammalian ancestors during the adaption of nocturnal lifestyle [61]. This may explain why the S-cones but not the green-cones are alternatively increased in the Nrl and Nr2e3 knockout mice [10,11,32,37].

Through single-cell RNA-seq, we further demonstrated the existence of different rod subtypes and revealed their signature developmental and gene expression patterns (Fig 7 and S1 Table). These findings explain the unique retinal phenotype observed in the nrl knockout zebrafish and suggest that zebrafish rods are derived from two different precursors under the control of regulatory genes, such as nrl and mafba. Recently, scRNA-seq has emerged as a powerful technique to study retinal development and degeneration under normal or pathological conditions [62]. This study also reported the cell compositions and cell-type-specific gene expression profiles in WT and nrl knockout retinas. Using these valuable data, we successfully identified the mafba gene as a potential driving factor for the formation of the nrl-independent rods. This assumption was subsequently validated in the nrl and mafba double-knockout zebrafish model. Moreover, we identified a cone-specific nuclear receptor gene, rorcb (RAR-related orphan receptor C b), to be up-regulated in the nrl knockout retinas (Figs 5A and 8A). Whether rorcb is responsible for the over-growth of green cones in the nrl knockout zebrafish needs to be determined in future studies.

In summary, we comprehensively characterized the retinal phenotype of nrl knockout zebrafish at the cellular and molecular levels and discovered novel roles of nrl and mafba in rod genesis, thereby contributing to the derivation of a more accurate working model for the photoreceptor development in zebrafish. Our findings may promote further studies in the fields of photoreceptor development and evolution, as well as retinal degeneration and regeneration in zebrafish.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009841

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