1. Neuroscience

cxcl18b-defined transitional state-specific nitric oxide drives injury-induced Müller glia cell-cycle re-entry in the zebrafish retina

  1. Aojun Ye  Is a corresponding author
  2. Shuguang Yu
  3. Meng Du
  4. Dongming Zhou
  5. Jie He  Is a corresponding author
  6. Chang Chen  Is a corresponding author
  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, China
  2. University of Chinese Academy of Sciences, China
  3. Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, China
  4. Tianjin Medical University, China
https://doi.org/10.7554/eLife.106274.3
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  1. Aojun Ye
  2. Shuguang Yu
  3. Meng Du
  4. Dongming Zhou
  5. Jie He
  6. Chang Chen
(2026)
cxcl18b-defined transitional state-specific nitric oxide drives injury-induced Müller glia cell-cycle re-entry in the zebrafish retina
eLife 14:RP106274.
https://doi.org/10.7554/eLife.106274.3
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7 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
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Single-cell RNA-sequencing (scRNA-seq) reveals injury-induced cxcl18b-defined Müller glia (MG) transitional states.

(A) Schematic showing the experimental procedure: 5 consecutive days of metronidazole (MTZ) treatment in Tg(lws2: nfsb-mCherry x mpeg1: GFP) fish to ablate green or red (G/R) cone, starting at 6 days post-fertilization (dpf) and continuing until 11 dpf. The MTZ solution was refreshed every 24 hr, followed by fish fixation for further immunostaining. (B–D) Quantitative plots showing the dynamic changes in the number of G/R cone (B), recruited microglia (C), and proliferative MG (PCNA+) (D) at different time points after MTZ treatment (uninjured: collected retina number n=14; 24 hr post-injury [hpi]: n=10; 48 hpi: n=10; 72 hpi: n=11; 96 hpi: n=12; 120 hpi: n=16; mean ± SEM; ****p<0.0001, ***p<0.001, **p<0.01, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (E) Representative images showing microglia recruitment (mpeg1: GFP, green), G/R cone ablation (lws2: nfsb-mCherry, red), and injury-induced MG proliferation (PCNA, white) in uninjured (E1) and 72 hpi (E2) retinas. The high-magnification images of the boxed area (E3– E4). Scale bars: 20 μm (E1, E2), 10 μm (E3), 2  μm (E4). (F) The UMAP plot of 4172 MG cells was sorted with an increased proportion in response to the G/R cone ablation. Cells were further aggregated into 10 clusters based on previously published scRNA-seq data (Krylov et al., 2023). (G) Pseudo-time developmental trajectory of MG states identified by Monocle2 analysis shows a main developmental branch originating from Cluster 4 (cx43+), which diverges into two sub-branches: Cluster 0 and Clusters 5/3/6 (pcna+). (H) Violin plots showing the expression levels of key genes (cx43, glula, gfap, cxcl18b, ascl1α, mki67, pcna, and notch3) in the main developmental branch clusters, progressing from the most original MG states (Cluster 4) to transitional MG states (Cluster 1/2/5), and proliferative MG states (Cluster 3/6).

Figure 1—source data 1

Quantification of the number of green/red (G/R) cones, recruited microglia, and PCNA+ Müller glia (MG) in the zebrafish retina after injury.

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Figure 1—figure supplement 1
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Clusters with increased proportion are identified from the single-cell RNA-sequencing (scRNA-seq) data.

(A) Representative images showing microglia recruitment (green, indicated by Tg(mepg1: GFP)), green/red (G/R) cone ablation (red, Tg(lws2: nfsb-mCherry)), and injury-induced Müller glia (MG) proliferation (white, marked by PCNA) following the injury process from uninjured retina to 120 hr post-injury (hpi). Scale bars: 20 μm. (B) Representative images showing PCNA+ (white, immunostaining) cells co-expressed with BLBP (red, immunostaining) at 72 hpi. Scale bars: 20 μm (B1– B3) and 5 μm (B4). (C) UMAP plot showing 5932 cells from uninjured retinas (blue) and 3999 cells from 72 hpi (red) retinas were obtained, which were further aggregated into 13 clusters. (D) Percentage of MGs from uninjured and 72 hpi retinas in each cluster. Eight clusters (6, 5, 9, 11, 12, 3, 2, 10) were identified and showed an increased proportion for further analysis. (E) Dot plot showing the expression levels of marker genes for quiescent MG (qMG), reactive MG (rMG), proliferative MG (pMG), and differentiated MG (dMG). The average expression levels of these genes for all cells in each cluster are coded by the gray level. The percentage of cells expressing each gene within each cluster is coded by dot size.

Figure 2 with 1 supplement
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Clonal analysis reveals the proliferative Müller glia (MG) mostly originated from cxcl18b+ MG transitional states.

(A) Representative images show dynamic expression of cxcl18b (red, in situ hybridization) and PCNA (white, immunostaining) in Tg(lws2: nfsb-mCherry) retina at different time points following the green/red (G/R) cone ablation. Scale bars: 20 μm. (B) Quantitative plots showing the number of cxcl18b+ (red curve, significance shown above the curve) and PCNA+ MG (blue curve, significance shown below the curve) in uninjured (n=11) and injured retina at 24 hr post-injury (hpi) (n=10), 48 hpi (n=12), 72 hpi (n=11), 96 hpi (n=7), and 120 hpi (n=5). Each injured time point was compared to the uninjured retina (mean ± SEM; ****p<0.0001, **p<0.01, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (C) Schematic diagram of the cxcl18b promoter was used to construct the reporter fish line Tg(cxcl18b: GFP) and clonal analysis fish line Tg(cxcl18b: Cre-vmhc-mCherry:: ef1α: loxP-DsRed-loxP-EGFP; lws2: nfsb-mCherry). (D) Immunostaining of injury-induced cxcl18b+ (green, indicated by Tg(cxcl18b: GFP)) and proliferative (PCNA+, white) MG showing overlapping in the central retina area (yellow arrows, GFP+/PCNA+ MG) at 48 hpi. The high-magnification images of the boxed area (D3'– D3'''). The area of the retina is labeled with a dashed line, and each layer structure is labeled with dashed lines and marked with the outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), and lens. Scale bars: 20 μm (D1– D2) and 5 μm (D3'– D3'''). (E) Quantitative plots showing the number of cxcl18b+ MG (red) and proliferative MG (PCNA+, blue) in (D1) uninjured (n=4) and (D2) 48 hpi retina (n=7) (mean ± SEM; ****p<0.0001, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test). (F) Clonal analysis of injury-induced cxcl18b+ MG in transgenic fish line Tg(cxcl18b: Cre-vmhc: mCherry:: ef1α: loxP-DsRed-loxP-EGFP:: lws2: nfsb-mCherry) at 72 hpi retina showing overlapping between proliferative (PCNA+, white) MG with cxcl18b+ (green, yellow arrows). The high-magnification images of the boxed area (F3'– F3'''). Scale bars: 20 μm (F1– F2) and 5 μm (F3'– F3'''). (G) Quantitative analysis at 72 hpi shows no significance in the number of proliferative MG (PCNA+, blue) and double-positive (PCNA+/cxcl18b+, red) MG (n=14; mean ± SEM; ns, p>0.05; unpaired t-test) in (F), with 93±2% of PCNA+ MG also being cxcl18b+. (H) Representative images show that not all mature MG stained with glutamate synthase (GS+, magenta) are cxcl18b+ (green, labeled by cxcl18b: Cre) in the central retinal area (white dashed lines identified a 45° angular region originating from the optic nerve). The high-magnification images of the boxed area (H2'– H2'''). Scale bars: 20 μm (H1) and 5 μm (H2'– H2'''). (I) Quantification of GS+/cxcl18b: Cre+ double-positive (blue, yellow arrows in H) and GS+/cxcl18b: Cre- single-positive (red, open yellow arrowheads in H) MG (n=14, mean ± SEM; *p<0.05; one-way ANOVA followed by Tukey’s HSD test), and the proportion of cxcl18b: Cre+ or – MG within the total population of mature (GS+) MG in the central retina. (J1– J3) Representative images showing injury-induced cxcl18b+ MG (green) in Tg(lws2: nfsb-mCherry) cross with Tg(cxcl18b: GFP) fish retina treated with dexamethasone (Dex) or DMSO at 72 hpi. Scale bars: 20 μm. (K – M) Quantitative plots showing the number of cxcl18b+ MG (72 hpi: n=14; DMSO: n=6, Dex: n=10) in J1-J3; recruited microglia in Figure 2—figure supplement 1J1–J3 and proliferative MG in Figure 2—figure supplement 1J4–J6 (72 hpi: n=9; DMSO: n=7, Dex: n=14) in Tg(mpeg1: GFP; lws2: nfsb-mCherry) retinas after DMSO or Dex treatment at 72 hpi (mean ± SEM; ****p<0.0001, ***p<0.001, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test).

Figure 2—source data 1

Quantitative analysis of cxcl18b in situ hybridization signal and PCNA+ Müller glia (MG) in uninjured and injured retinas at the indicated time points.

https://cdn.elifesciences.org/articles/106274/elife-106274-fig2-data1-v1.xlsx
Figure 2—source data 2

Quantification of the number of cxcl18b+ and PCNA+ Müller glia (MG) in the uninjured and 48 hr post-injury (hpi) zebrafish retinas from Tg(cxcl18b: GFP) fish.

https://cdn.elifesciences.org/articles/106274/elife-106274-fig2-data2-v1.xlsx
Figure 2—source data 3

Quantification of the number of cxcl18b+ and PCNA+/cxcl18b+ double-positive Müller glia (MG) in injured zebrafish retinas in the lineage-tracing experiment.

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Figure 2—source data 4

Quantification of GS+/cxcl18b: Cre+ and GS+/cxcl18b: Cre⁻ Müller glia (MG) in the central retina region.

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Figure 2—source data 5

Quantification of cxcl18+ Müller glia (MG), microglia, and PCNA+ MG in the immunosuppression experiment.

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Figure 2—figure supplement 1
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Gene description of cxcl18b does not reduce Müller glia (MG) proliferation.

(A–A3) In situ hybridization images show injury-induced cxcl18b (red, marked by in situ hybridization) with MG-like morphology, cell-specific expression in the BLBP+ cells (green, labeled by immunostaining) at 24 hr post-injury (hpi) in the transgenic fish line Tg(lws2: nfsb-mCherry) retina. In these clones, cxcl18b+ MG are red+/BLBP+ cells (yellow arrowheads). The high-magnification images of the boxed area (A1– A3). The area of the retina structure is labeled with a dashed line, and each layer structure is labeled with dashed lines and marked with the outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), and lens. Scale bars: 20 μm (A) and 5 μm (A1– A3). (B – B3) Representative images showing cxcl18b (red, in situ hybridization) co-expressed with PCNA (white, immunostaining) at 72 hpi. In these clones, cxcl18b+ MG was identified as red+/PCNA+ cells (yellow arrowheads). The high-magnification images of the boxed area (B1– B3). Scale bars: 20 μm (B) and 5 μm (B1– B3). (C) Schematic showing the design of the cxcl18b reporter fish line and the Cre-loxP transgenic fish line used for clone analysis. The cxcl18b promoter drives GFP or Cre expression, with the heart labeled by mCherry under the vmhc promoter for fish screening. (D – E3) Representative images showing cxcl18b expression labeled by Tg(cxcl18b: GFP) (green) in (D–D3) at 48 hpi, or three transgenic fish Tg(cxcl18b: Cre-vmhc: mCherry:: ef1α: loxP-DsRed-loxP-EGFP:: lws2: nfsb-mCherry) (green) in (E – E3) at 24 hpi fish retina, merged with cxcl18b in situ hybridization signal (red) (D – E3). The high-magnification images of the boxed area (D1– D3, E1– E3). Scale bars: 20 μm (D, E) and 5 μm (D1– D3, E1– E3). (F) Schematic showing the two sgRNA target sites of the cxcl18b genome. (G) Summary table showing the indel and knockout efficiency of the two sgRNAs targeting cxcl18b. (H) Representative images of proliferative MG (PCNA+, white) at 72 hpi in Tg(lws2: nfsb-mCherry) fish retina with cxcl18b disruption. Scale bars: 20 μm. (I) Quantitative plots showing the number of proliferative MG (PCNA+) at 72 hpi in (H). In total, retinas were collected from wild-type (WT, n=11), scramble sgRNA-injected (scrambled, n=7), and cxcl18b sgRNA-injected (cxcl18b KO, n=7) (mean ± SEM; ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (J1– J6) Representative images showing injury-induced microglia recruitment (green, marked by Tg(mpeg1: GFP)) (J1– J3), and proliferative MG (white, marked by PCNA) (J4– J6) after dexamethasone (Dex) or DMSO treatment in the Tg(mpeg1: GFP x lws2: nfsb-mCherry) fish retina at 72 hpi. Scale bars: 20 μm.

Figure 2—figure supplement 1—source data 1

Quantification of PCNA+ Müller glia (MG) in the injured retinas under control and cxcl18b disruption conditions.

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Figure 3 with 1 supplement
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The cxcl18b-defined Müller glia (MG) transitional states recapitulate the developmental states of retinal stem cells (RSCs) in the ciliary marginal zone (CMZ).

(A, C) UMAP plots display 5368 retinal progenitor cells (RPCs) at 24 hr post-fertilization (hpf) and 4625 CMZ progenitor cells at 14 days post-fertilization (dpf). Clusters are indicated by their cluster-specific marker genes based on previously published single-cell RNA-sequencing (scRNA-seq) data (Xu et al., 2020). (B, D) UMAP plots showing expression of fabp11a, cxcl18b, and col15α1b at r24-hpf RPCs (embryonic states) (B) and r14-dpf CMZ-progenitors (postembryonic states) (D). (E – H3) In situ hybridization images showing the expression of fabp11a (green), col15α1b (white), two putative markers for postembryonic RSCs, and cxcl18b at 30 hpf (E – F3) and 14 dpf (G – H3) retina. The high-magnification images of the boxed area (F – F3, H – H3). The area of these three in situ signal trouble positives is labeled with a dashed yellow line. Scale bars: 20 μm (E, G) and 3 μm (F – F3, H – H3).

Figure 3—figure supplement 1
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UMAP plots show the expression of cluster-specific marker genes utilized to identify developmental states.

(A, B) UMAP plots showing the expression pattern of embryonic developmental stage cluster-specific marker genes (npm1a, her9, her4.2, fabp7a, dla, and atoh7) in (A) and postembryonic developmental stage marker genes (her4.2, atoh7, vsx1, mafba, otx5, and rem1) in (B). (C) UMAP plots showing the expression pattern of fabp11a, cxcl18b, and col15α1b at Müller glia (MG) clusters with increased population after green/red (G/R) cone ablation at 72 hr post-injury (hpi) in Figure 1F. (D) Representative images showing the cxcl18b expression (green, marked by Tg(cxcl18b: GFP)) are located in the most peripheral region of the ciliary marginal zone (CMZ) (white, marked by PCNA) at 5 days post-fertilization (dpf). The high-magnification images of the boxed area (D1–D3). Scale bars: 20 μm (D) and 5 μm (D1– D3).

Figure 4 with 2 supplements
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The nitric oxide metabolic pathway regulates injury-induced Müller glia (MG) proliferation.

(A) Representative images of microglia recruitment (green, marked by Tg(mpeg1: GFP)), green/red (G/R) cone ablation (red, marked by Tg(lws2: nfsb-mCherry)) and proliferative MG (white, marked by PCNA+) at 72 hr post-injury (hpi) with nitric oxide metabolism pathway genes disruption (nos1/nos2a/nos2b/gsnor). Scale bars: 20 μm. (B) Quantitative analysis of the number of proliferative MG (PCNA+) at 72 hpi in (A). In total, we collected 11 retinas for wild-type (WT) (n=11), scramble sgRNA-injection (n=7), nos1 sgRNA-injection (n=23), nos2a sgRNA-injection (n=15), nos2b sgRNA-injection (n=22), and gsnor sgRNA-injection (n=13) (mean  ± SEM; ****p<0.0001, **p<0.01, *p<0.05, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (C) Schematic showing the experimental procedure of nitric oxide synthase (Nos) inhibitors injection or NO scavengers treatment in Tg(lws2: nfsb-mCherry × mpeg1: GFP) retina starting from 5 days post-fertilization (dpf) for 5 consecutive days to 10 dpf with 3 consecutive days of metronidazole (MTZ) treatment for G/R cone ablation from 7 dpf to 10 dpf. Nos inhibitors, NO scavengers, and MTZ solution were refreshed every 24 hr, and fish fixation was at 10 dpf for further immunostaining. (D) Representative images of microglial recruitment (green, marked by Tg(mpeg1: GFP)), G/R cone ablation (red, marked by Tg(lws2: nfsb-mCherry)), and proliferative MG (white, marked by PCNA+) at 72 hpi following L-NMMA (10 mM), L-NAME (10 mM), 1400W (200 nM) intraocular injection, and PBS as control, or C-PTIO (10 mM) treatment. Scale bars: 20 μm. (E) Quantitative plots showing the number of proliferative (PCNA+) MG at 72 hpi in (D). Retinas analyzed WT (n=11), PBS-injected (n=16), L_NMMA-injected (n=12), L_NAME-injected (n=12), 1400W-injected (n=10), and C-PTIO treatment (n=27) (mean ± SEM; ****p<0.0001, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (F–G) Representative images of proliferative MG (PCNA+, white) and G/R cone ablation (marked by Tg(lws2: nfsb-mCherry), red) at 72 hpi in heterozygous (nos1+/-, nos2a+/-, nos2b+/-, gsnor+/-) (F) and homozygous mutants (G) of nitric oxide metabolism pathway genes (nos1-/-, nos2a-/-, nos2b-/-, gsnor-/-). Scale bars: 20  μm. (H–I) Quantitative plots showing the number of proliferative MG (white, PCNA+) at 72 hpi in nos and gsnor mutant fish. In heterozygous (H), analyzed retinas include WT (n=14), nos1+/- (n=19), nos2a+/- (n=13), nos2b+/- (n=20), gsnor+/- (n=12). For homozygous (I), analyzed retinas include nos1-/- (n=18), nos2a-/- (n=20), nos2b-/- (n=20), gsnor-/- (n=27) (mean ± SEM; ****p<0.0001, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (J–K) Quantitative plots showing the number of proliferative MG (white, PCNA+) (J) and photoreceptor cells remain (K) at 72 hpi in nos2b hetero- or homozygous mutants (mean ± SEM; ****p<0.0001, ***p<0.001, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test).

Figure 4—source data 1

Quantitative analysis of PCNA+ Müller glia (MG) in the Nos metabolism gene disruption experiment after retinal injury.

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Figure 4—source data 2

Quantitative analysis of PCNA+ Müller glia (MG) in the Nos inhibitor or NO scavenger injection experiment after injury.

https://cdn.elifesciences.org/articles/106274/elife-106274-fig4-data2-v1.xlsx
Figure 4—source data 3

Quantification of the number of PCNA+ Müller glia (MG) and photoreceptor cells in the retinas of fish with Nos mutations.

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Figure 4—figure supplement 1
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Genetic disruption of the nitric oxide (NO) pathway modulates green/red (G/R) cone ablation and microglial recruitment.

(A) Dot plot showing the redox gene expression profiles of regulating glutathione (GSH), hydrogen peroxide (H2O2), nicotinamide adenine dinucleotide phosphate (NADPH), NO, metal ion, and sulfhydryl group (-SH) in the uninjured and cxcl18b+ Müller glia (MG) in 72 hr post-injury (hpi) single-cell RNA-sequencing (scRNA-seq) data (Krylov et al., 2023). The average expression levels of these genes for all cells in each cluster were coded by the gray level. The percentage of cells expressing each gene within each cluster was coded by dot size. (B) Schematic showing the two sgRNA target sites of the NO metabolism pathway genome (nos1/nos2a/nos2b/gsnor). (C) Summary table showing the indel and knockout efficiency of the two sgRNAs targeting nos1/nos2a/nos2b/gsnor. (D) Representative images of microglial recruitment (marked by Tg(mpeg1: GFP), green) and G/R cone ablation (marked by Tg(lws2: nfsb-mCherry), red) at 72 hpi with nos1/nos2a/nos2b/gsnor disruption, with scrambled sgRNA-injected as the control. Scale bars: 20 μm. (E–F) Quantitative plots showing the number of recruited microglia (GFP+) (D) and G/R cone ablation (mCherry+) (E) at 72 hpi with nos1/nos2a/nos2b/gsnor disruption. In total, we collected 11 retinas for wild-type (WT) (n=11), scrambled sgRNA-injected (n=7), nos1 sgRNA-injected (n=10), nos2a sgRNA-injected (n=6), nos2b sgRNA-injected (n=12), and gsnor sgRNA-injected (n=13) (mean ± SEM; *p<0.05; ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test).

Figure 4—figure supplement 2
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The genotype of nitric oxide metabolism pathway mutants.

(A) Sequence alignment of wild-type (WT) nitric oxide metabolism pathway genes (nos1/nos2a/nos2b/gsnor) and their CRISPR-cas9-induced mutant alleles. (B) Summary table showing the premature stop codon and unknown polypeptide sides of each mutant.

Figure 5 with 1 supplement
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Real-time quantitative PCR (RT-qPCR) analysis reveals nos2b cell-specific expression in the injury-induced cxcl18b+ Müller glia (MG).

(A) Schematic showing the workflow for isolating and enriching for three post-injury MG populations (72 hr post-injury [hpi] MG, 72 hpi PCNA+ MG, and 72 hpi cxcl18b+ MG) and three control groups (uninjured MG, 72 hpi retinal cells other than MG, and 72 hpi green/red [G/R] cones) using fluorescence-activated cell sorting (FACS). (B–C) RT-qPCR analysis of nos1, nos2a, nos2b, and gsnor expression in different cell populations. Expression levels are shown relative to 72 hpi G/R cones (B) and other 72 hpi retinal cell types (C). A total of six independent replicates were performed for cell population enrichment and cDNA template preparation (n=6, mean ± SEM; ****p<0.0001, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test). (D) RT-qPCR analysis comparing nos1, nos2a, nos2b, and gsnor expression in distinct MG states relative to uninjured MG (repeats n=7 in cxcl18b+ MG; n=5 in 72 hpi MG; and repeats n=6 in PCNA+ MG; n=6 in the uninjured retina; mean ± SEM; ****p<0.0001, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test).

Figure 5—source data 1

Real-time quantitative PCR (RT-qPCR) analysis of nos1, nos2a, nos2b, and gsnor expression across retinal cell populations and distinct Müller glia (MG) states.

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Figure 5—figure supplement 1
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In situ hybridization reveals nos2b cell-specific expression in the cxcl18b-defined transitional Müller glia (MG) states.

(A–A3) Representative in situ hybridization images showing nos2b expression (red, in situ hybridization) cell-specific in the injury-induced cxcl18b+ MG (GFP+, green) in Tg(lws2: nfsb-mCherry × cxcl18b: GFP) fish retina at 72 hr post-injury (hpi). Clones with green/red/white trouble-positive are cxcl18b+/nos2b+/PCNA+ MG cells (yellow arrowheads). The high-magnification images of the boxed area (C–C3). Scale bars: 20  μm (A, B) and 5 μm (C1–C3). (D–E) Real-time quantitative PCR (RT-qPCR) analysis of glula (pink bar) and glulb (green bar) expression in different cell populations relative to 72 hpi green/red (G/R) cone (D) or other retinal cell types (E). The significance of 72 hpi G/R cone as control is noted by * for comparisons with 72 hpi G/R cones and # for comparisons with uninjured MG. In total, we do 6 independent replicates to enrich the different cell populations and get the cDNA templates (mean ± SEM; **** and ####p<0.0001, **p<0.001, **p<0.01, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test). (F) RT-qPCR analysis of cxcl18b (blue bar) and pcna (orange bar) expression in different MG states (mean ± SEM; ****p<0.0001, **p<0.01, *p<0.05, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test).

Figure 5—figure supplement 1—source data 1

Real-time quantitative PCR (RT-qPCR) analysis of glula, glulb, cxcl18b, and pcna expression across retinal cell populations and Müller glia (MG) states.

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Figure 6 with 1 supplement
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Nitric oxide (NO) produced by nos2b in cxcl18b+ Müller glia (MG) regulates injury-induced proliferation.

(A–C) Representative images showing the adenovirus-mediated infection (green, indicated by Y2-GFP) specifically target MG (red, GS staining) in the zebrafish retina. The virus was intraocularly injected into the right eye of Tg(lws2: nfsb-mCherry x ef1α: loxP-DsRed-loxP-EGFP) fish (A2, C), with the left eye as a wild-type (WT) control (A1). The high-magnification images of the boxed area (B–B3). Scale bars: 20 μm (A1, A2) and 5 μm (B–B3). (D) Schematic showing the design of the cxcl18b+ MG-specific nos2b knockout system. The viral construct consists of three plasmids: (1) gal4 expression driven by the cxcl18b promoter; (2) UAS-derived Cas9 and Cre elements, and (3) U6 promoters driving two sgRNAs targeting nos2b, with a non-targeting sgRNA as the control. (E) Schematic showing the procedure of injury process and intraocular viral injection in Tg(lws2: nfsb-mCherry × ef1α: loxP-DsRed-loxP-EGFP) fish. (F–G) Representative images showing proliferative MG (PCNA+, white) with cxcl18b+ MG-specific knockout nos2b in (G) and control in (F), (GFP+, green, yellow arrows) are defined as virus-infected clones. Upper panels show WT retina (no virus injected). Bottom panels show retinas injected with the virus (two sgRNA targets as nos2b knockout and without sgRNA targets as control). Scale bars: 20 μm. (H) Quantification of proliferative (PCNA+/GFP+, red bars) and non-proliferative (PCNA-/GFP+, gray bars) MG clones in (F). For control, ~75% of virus-infected clones entered the cell cycle (PCNA+; red bars), with 90/120 clones analyzed across 8 independent experiments (n=8). For nos2b knockout clones, ~23% entered the cell cycle (PCNA+; blue bars), with 22/103 clones analyzed across 6 independent experiments (n=6) (mean ± SEM; ****p<0.0001; two-way ANOVA followed by Tukey’s HSD test).

Figure 6—source data 1

Quantification of proliferative (PCNA+/GFP+) and non-proliferative (PCNA⁻/GFP+) Müller glia (MG) clones in control and nos2b MG-specific knockout conditions.

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Figure 6—figure supplement 1
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Analysis of cell-specific knockout efficiency for nos2b in virus-infected clones.

(A) Schematic showing the cell-specific gene knockout system design. Injury-induced cxcl18b promoter directs the expression of gal4, which in combination with the UAS element, drives the cell-specific expression of Cas9 and Cre. The U6 promoter enables the broad expression of sgRNAs targeting nos2b, and Cas9 mediates gene knockout within the targeted cells through these sgRNAs. The ef1α promoter controls the loxP-DsRed-loxP-EGFP reporter system, allowing for the switch from DsRed to EGFP upon Cas9 and Cre activity, and GFP+ Müller glia (MG) clones are identified with successful nos2b knockout. This system operates with high specificity via double adenovirus infection. (B) Schematic showing the procedure of virus-infected clones sorted and enriched by fluorescence-activated cell sorting (FACS) for further knockout efficiency analysis. (C) Representative gel electrophoresis image showing nos2b deletions in knockout cells compared to controls. Single-virus injections were used to assess Cre system leakage. While minimal Cre system leakage was observed, it did not result in detectable nos2b knockout. (D) Summary table showing the knockout efficiency of each virus-injected clone.

Figure 6—figure supplement 1—source data 1

PDF file containing original DNA gel for Figure 6—figure supplement 1C, indicating the relevant bands.

https://cdn.elifesciences.org/articles/106274/elife-106274-fig6-figsupp1-data1-v1.zip
Figure 6—figure supplement 1—source data 2

Original files for DNA gel analysis displayed in Figure 6—figure supplement 1C.

https://cdn.elifesciences.org/articles/106274/elife-106274-fig6-figsupp1-data2-v1.zip
Figure 7
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Nitric oxide (NO) regulates Müller glia (MG) proliferation by suppressing Notch signaling.

(A) Dot plot showing the Notch signaling-related gene expression in different MG states. The average expression levels of these genes for all cells in each cluster are coded by the gray level. The percentage of cells expressing each gene within each cluster is coded by dot size. (B) Representative images showing the dynamic changes of Notch signaling activity (green, indicated by Tg(Tp1: EGFP)) and proliferative MG (white, PCNA+) following injury with nitric oxide (NO) blockade using C-PTIO. Scale bars: 20 μm. (C–D) Quantitative plots showing the number of Notch activation (tp1: GFP+ MG) in (C) and proliferative MG (PCNA+ MG) in (D) at different time points post-green/red (G/R) cone ablation. We collected uninjured retina (24 hr post-injury [hpi], n=4; 48 hpi, n=4; 72 hpi, n=3), injured retina (24 hpi, n=9; 48 hpi, n=4; 72 hpi, n=6), and retina treated with C-PTIO (24 hpi, n=6; 48 hpi, n=5; 72 hpi, n=7) (mean ± SEM; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test).

Figure 7—source data 1

Quantification of Notch-activated (tp1: GFP+) Müller glia (MG) and PCNA+ MG at different time points under uninjured, injured, and C-PTIO-treated conditions.

https://cdn.elifesciences.org/articles/106274/elife-106274-fig7-data1-v1.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Danio rerio) Wild typeDr. WilliamZIRC_ZL1AB
Strain, strain background (Danio rerio) lws2: nfsb-mCherryKrylov et al., 2023ZDB-TGCONSTRCT-230530-2Tg(opn1lws2: nfsb-mCherry)uom3
Strain, strain background (Danio rerio) her4.1: dRFPYeo et al., 2007ZDB-TGCONSTRCT-070612-2Tg(her4.1: dRFP)
Strain, strain background (Danio rerio) gfap: EGFPBernardos and Raymond, 2006ZDB-FISH-150901-29,307Tg(gfap: EGFP)
Strain, strain background (Danio rerio) mpeg1: GFPEllett et al., 2011ZDB-TGCONSTRCT-170801–5Tg(mpeg1: GFP)
Strain, strain background (Danio rerio) Tp1bglob: EGFPYu and He, 2019ZDB-TGCONSTRCT-090625-1Tg(Tp1bglob: EGFP)
Strain, strain background (Danio rerio) pcna: GFPXu et al., 2020ZDB-LAB-070129-2Tg(pcna: GFP)
Strain, strain background (Danio rerio) ef1α: loxP-DsRed-loxP-EGFPThis manuscriptTg(ef1α: loxP-DsRed-loxP-EGFP)
Strain, strain background (Danio rerio) cxcl18b: Cre-vmhc: ECFPThis manuscriptTg(cxcl18b: Cre-vmhc: ECFP; ef1α: loxP-DsRed-loxP-EGFP; lws2: nfsb-mCherry)
Strain, strain background (Danio rerio) cxcl18b: GFPThis manuscriptTg(cxcl18b: GFP)
AntibodyMouse monoclonal anti-PCNAAbcamCat#Ab29; RRID:AB_303394IF(1:500)
AntibodyRabbit polyclonal anti-BLBPAbcamCat#ab32423; RRID:AB_880078IF(1:1000)
AntibodyMouse monoclonal anti-Glutamine SynthetaseBD BiosciencesCat# 610518; RRID:AB_397880IF(1:1000)
AntibodyChicken monoclonal anti-GFPAbcamCat# ab13970; RRID:AB_300798IF(1:2000)
AntibodyRabbit polyclonal anti-GFPtagRabbit polyclonal anti-GFPtagCat#50430–2-AP; RRID:AB_11042881IF(1:500)
AntibodyRabbit polyclonal anti-DsRed2Takara BioCat#632496; RRID:AB_10013483IF(1:1000)
AntibodyAlexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L)Yeasen BiotechCat# 33206ES; RRID:AB_3662603IF(1:1000)
AntibodyAlexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L)Yeasen BiotechCat# 34206ES60; RRID:AB_2909605IF(1:1000)
AntibodyAlexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L)Jackson ImmunoResearch LabsCat# 703-545-155; RRID:AB_2340375IF(1:1000)
AntibodyAlexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L)Yeasen BiotechCat# 34112ES; RRID:AB_3661960IF(1:1000)
AntibodyAlexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L)Yeasen BiotechCat# 33112ES; RRID:AB_3661961IF(1:1000)
AntibodyAlexa Fluor 647-AffiniPure Goat Anti-Mouse IgG +IgM (H+L)Jackson ImmunoResearch LabsCat# 115-605-044; RRID:AB_2338906IF(1:1000)
Recombinant DNA reagentpTol2-cxcl18b: GFPThis manuscriptWe made this plasmid by ligating the cxcl18b promoter and GFP element
Recombinant DNA reagentpTol2-cxcl18b: Cre-vmhc: mCherryThis manuscriptWe made this plasmid by ligating the cxcl18b promoter and Cre element
Recombinant DNA reagentpTol2-cxcl18b: gal4FFThis manuscriptWe made this plasmid by ligating the cxcl18b promoter and gal4FF element
Recombinant DNA reagentpUAS: Cas9T2ACre; U6: sgRNA1; U6: sgRNA2Di Donato et al., 2016Addgene plasmid #74010; RRID:Addgene_74010 
Recombinant DNA reagentpTol2-UAS: Cas9-T2A-Cre-U6: nos2b sgRNA1; U6: nos2b sgRNA2This manuscriptWe made this plasmid by inserting two sgRNAs of nos2b in 10xUAS backbone
Commercial assay or kitMEGAscriptTM T7 High Yield Transcription KitInvitrogenCat# AM1334 
Commercial assay or kitClonExpress MultiS One Step Cloning KitVazymeCat# C113-02 
Commercial assay or kitDIG RNA labeling kitRocheCat# 11277073910 
Chemical compound, drugN(ω)-nitro-L-arginine methyl esterSigma-AldrichCat# N5751-1G10 mM
Chemical compound, drugN(ω)-methyl-L-arginine acetate saltSigma-AldrichCat# M7033-5MG10 mM
Chemical compound, drug1400W dihydrochlorideMedChemExpressCat# HY-18730200 nM
Chemical compound, drugPhenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxideSigma-AldrichCat# P5084-25MG10 mM
Chemical compound, drugDexamethasoneSigma-AldrichCat# D175610 mM
Chemical compound, drugMetronidazoleSigma-AldrichCat# M3761-100G10 mM
Software, algorithmFV10-ASW 4.0 ViewerOlympushttps://olympus-global.comAnalysis image
Software, algorithmGraphPad Prism V 9.0.0GraphPad Softwarehttps://graphpad.comData analysis
Software, algorithmCell Ranger Single Cell Software Suite (v2.1.0) 10x Genomicshttps://support.10xgenomics.comscRNA-seq data analysis
Software, algorithmRStudioRStudio IDEhttps://posit.co/scRNA-seq data analysis
Software, algorithmR 4.4.1R-projecthttps://www.r-project.org/scRNA-seq data analysis
Software, algorithmSeuratSatijalabhttps://satijalab.org/seurat/scRNA-seq data analysis

Additional files

Supplementary file 1

sgRNA sequences and genotyping primers, related to STAR methods.

https://cdn.elifesciences.org/articles/106274/elife-106274-supp1-v1.docx
Supplementary file 2

Plasmid construction primer sequences, related to STAR methods.

https://cdn.elifesciences.org/articles/106274/elife-106274-supp2-v1.docx
Supplementary file 3

qPCR primer sequences, related to STAR methods.

https://cdn.elifesciences.org/articles/106274/elife-106274-supp3-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/106274/elife-106274-mdarchecklist1-v1.pdf

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  1. Aojun Ye
  2. Shuguang Yu
  3. Meng Du
  4. Dongming Zhou
  5. Jie He
  6. Chang Chen
(2026)
cxcl18b-defined transitional state-specific nitric oxide drives injury-induced Müller glia cell-cycle re-entry in the zebrafish retina
eLife 14:RP106274.
https://doi.org/10.7554/eLife.106274.3