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.2022 Mar 4;18(3):e1009841.
doi: 10.1371/journal.pgen.1009841. eCollection 2022 Mar.

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

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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 et al. PLoS Genet..

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Knocking outnrl diminishes but does not eradicate the rod population in zebrafish.
(A) The gene structure of zebrafishnrl and the CRISPR-Cas9 target site used for knocking outnrl 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 homozygousnrl del8 mutation (c.230_237del8). (C) Thenrl mRNA levels in thenrl-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 andnrl-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.
Fig 2
Fig 2. Reduced density of the rod outer segments in thenrl-KO zebrafish.
(A) The rod outer segments were visualized via immunostaining on the flattened whole-mount retinas from WT andnrl-KO zebrafish at 2 mpf using the anti-Rho antibody. The overall views of WT andnrl-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 thenrl knockout retinas, especially in the ventral retinal region. Scale bar: 50 μm.
Fig 3
Fig 3. Tracing the developmental processes of rods in WT andnrl-KO zebrafish.
Rods were labeled with EGFP by crossing the WT ornrl-KO zebrafish with the Tg(rho:EGFP) transgenic line. Fluorescence was observed every 2 days from 3 dpf. Representative images of WT andnrl-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 thenrl-KO retinas. Scale bar: 50 μm.
Fig 4
Fig 4. Distribution of the rods and expression of the rod opsin in the WT andnrl-KO retinas from 1 mpf to 3 mpf.
(A) The Tg(rho:EGFP) transgenic line was used to label rods with EGFP. Representative images of flattened whole-mount retinas from WT andnrl-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 andnrl-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.
Fig 5
Fig 5. Differentially expressed genes between WT andnrl-KO retinas.
(A) The volcano plot shows the 386 differentially expressed genes (196 up-regulated and 190 down-regulated) between WT andnrl-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 innrl-KO retinas shown as a heatmap. Most of the rod genes were down-regulated in thenrl-KO retinas.
Fig 6
Fig 6. Down-regulation of rod-specific genes and increased number of green cones in adultnrl-KO zebrafish.
(A) The protein levels of rod-specific (rho,gnat1, andgnb1) and cone-specific (gnat2 andgnb3) genes in WT andnrl-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 andnrl-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 andnrl-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.
Fig 7
Fig 7. Single-cell RNA-seq analysis in WT andnrl-KO retinas.
(A) tSNE visualization of the unsupervised cell clusters from 5-month-old WT andnrl-KO zebrafish. Left, the distribution of cell clusters between the WT andnrl-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 andnrl-KO zebrafish. (C) The cell compositions and proportions of photoreceptor sub-clusters in WT andnrl-KO retinas. (D) The heatmap shows the clustering pattern of the top 20 down-regulated and top 20 up-regulated genes in the WT andnrl-KO rods. Yellow, high expression; purple, low expression. (E) Mis-expression of green-cone opsin in a proportion of rods in thenrl-KO retinas. The dorsal retinal regions of WT andnrl-KO zebrafish at 3 mpf are shown. Green, EGFP-labeled rods. Red, immunofluorescence signals of the anti-Opn1mw antibody. The green fluorescent signal in thenrl-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.
Fig 8
Fig 8. Identification of themafba gene as a novel driving factor for the development ofnrl-independent rods.
(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 ofmafba was highly similar to those ofnrl andnr2e3. (B) The distribution of rods (labeled with EGFP) in thenrl-KO zebrafish carrying the WT, heterozygous, and homozygousmafba alleles at 9 dpf (left panel) and 15 dpf (right panel). Knocking outmafba further reduced the genesis of rods in thenrl-KO zebrafish. The dotted circles indicate the boundaries of the retinas. Scale bars: 100 μm. (C) The expression levels ofnrl,nr2e3, andmafba in thenrl-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.
Fig 9
Fig 9. Gene collinearity ofmafba among species at different evolutionary positions.
The genes nearbymafba 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). Themafba andmafbb genes appear to have originated from the ancestral genemafb through genome duplication specifically in teleost fishes.
Fig 10
Fig 10. Progressive degeneration and regeneration in thenrl-KO retinas.
(A) The TUNEL assay results revealed that multiple types of retinal cells, including rods, cones, RPE, and inner retinal cells, undergo apoptosis in thenrl-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 andnrl-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 innrl-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 thenrl-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.
Fig 11
Fig 11. Working model for the development of rod photoreceptors in the zebrafish.
There may be two types of rod precursors defined asnrl-dependent andnrl-independent in the zebrafish retinas. The former is responsible for the production of rods at embryonic stage, and knocking outnrl 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 ofnrl andmafba, these cells differentiate into rods. Knocking outnrl 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 bothnrl andmafba eliminates all rods, suggesting thatmafba also plays an important role in the fate determination of this type of rods (right panel).
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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.

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