Nitric Oxide Fuels Müller Glia Cell-Cycle Re-entry for Zebrafish Retina Regeneration

Zebrafish MG can respond to retinal injury by cell-cycle re-entry, a critical step evolutionarily absent from their mammalian counterparts but essential for neuron regeneration (Goldman, 2014; Powell et al., 2016). We created a zebrafish retinal injury model by crossing Tg(opn1lws2: nfsb-mCherry)uom3 (referred to as Tg(lws2: nfsb-mCherry)) with Tg(mpeg1: GFP) fish, in which the bacterial nitroreductase (NTR) enzyme was specifically expressed in G/R cone. We selectively ablated the G/R cone starting at 5 days post-fertilization (dpf) by a subsequent 120 hr of metronidazole (MTZ) exposure (Curado et al., 2007; Curado et al., 2008; Figure 1A, Figure 1—figure supplement 1A). G/R cone became significantly reduced in number since 48 hr post-injury (hpi) and was mostly depleted at 120 hpi (Figure 1B). Meanwhile, a number of microglia (marked by Tg(mpeg1: GFP)) migrated to the outer nuclear layer (ONL) as early as 48 hpi, peaked at 72 hpi, and began to reduce in number at 96 hpi and onward (Figure 1C). To confirm the identity of these proliferating cells, we performed BLBP immunostaining and observed that the PCNA+ cells were also BLBP+ (Figure 1—figure supplement 1B), indicating their MG origin. Notably, in response to G/R cone ablation, the proliferative MG population increased starting at 48 hpi, peaked at 72 hpi, and began to decline since 96 hpi and forward (Figure 1D). Considering the result that the number of proliferative MG peaked at 72 hpi, we focused on the 72 hpi time point for further exploration of MG proliferative behaviors following G/R cone ablation (Figure 1E). 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. https://cdn.elifesciences.org/articles/106274/elife-106274-fig1-data1-v1.xlsx By revisiting previously obtained scRNA-seq data of MG enriched from Tg(lws2: nfsb-mCherry) crossed with Tg(gfap: EGFP) and Tg(her4.1: dRFP) retina before and after G/R cone ablation at 72 hpi (Krylov et al., 2023), we selected 5932 and 3999 MG cells and their derived progenies from the uninjured and 72 hpi retina, respectively (details in Materials and methods; Figure 1—figure supplement 1C). By clustering these cells, we identified 13 clusters, 8 out of which (Clusters 2, 3, 5, 6, 9, 10, 11, and 12) with an increased proportion in response to the G/R cone ablation (Figure 1—figure supplement 1D). Subsequently, we re-clustered cells of these 8 clusters (695 cells of uninjured retinae and 3477 cells of 72 hpi retinae), aggregating into 10 new clusters. After the quality control procedure, we did not consider Clusters 7/8/9 due to their small populations with ribosomal, dendritic, and doublet features, resulting in 7 clusters for further analysis (details in Materials and methods; Figure 1F). We performed the pseudo-time trajectory analysis to reveal the progression of these MG clusters following the cone ablation (Figure 1G). Cluster 4 cells were highly expressing genes related to mature MG (glula, slc1a2b, apoeb, and rlbp1a) (Bernardos and Raymond, 2006; Raymond et al., 2006; Thummel et al., 2008; Yurco and Cameron, 2005), the quiescent state (cx43) (Dermietzel et al., 2000; Janssen-Bienhold et al., 1998), and major MG population marker (fgf24) (Krylov et al., 2023; Figure 1—figure supplement 1E). Furthermore, Cluster 4 began to express s100α10b and gfap, reactive state makers (Celotto et al., 2023; Hoang et al., 2020), in the injured retinae, but not in the uninjured retina (Figure 1—figure supplement 1E). Thus, we set Cluster 4 as the early transitional MG state. To examine the transition of these 7 MG clusters, the pseudo-time trajectory showed that the main developmental branch consisted of Cluster 4/1/2 and then became divergent into two sub-branches, including Clusters 0 and 5/3/6 (Figure 1G). In contrast to the sub-branch of Cluster 0, the sub-branch of Cluster 5/3/6 was highly expressing proliferative cell markers (pcna, mki67, and mcm2). Within the latter sub-branch, while Clusters 3 and 6 had the highest levels of proliferative cell markers, Cluster 6 began to express neuronal differentiation factors (otx5, crx, and pde6gb) (Abalo et al., 2020; Asaoka et al., 2014; Shen and Raymond, 2004; Figure 1—figure supplement 1E). Thus, we identified 6 major post-injury MG states, from the early transitional state (Cluster 4) to three transitional states (Cluster 1/2/5), to finally two proliferative states (Cluster 3/6). Remarkably, chemokine (C-X-C motif) ligand 18b (cxcl18b), an inflammatory chemokine, was uniquely expressed in three transitional states but largely absent from the early transitional Cluster 4 and two proliferative Cluster 3/6. Specifically, while the first cxcl18b+ transitional state (Cluster 1) was expressing cx43, a marker for MG quiescence (Dermietzel et al., 2000; Janssen-Bienhold et al., 1998), the last transitional state (Cluster 5) began to show a weak induction of ascl1α (Ramachandran et al., 2010). Immediately following this last transitional state, Cluster 3 started with high ascl1α expression and entered the proliferative state with the expression of pcna and mik67 (Figure 1H). Our analysis highlighted a new set of cxcl18b-defined MG transitional states preceding ascl1α induction, bridging MG from the most original quiescence state to injury-induced proliferation. To directly verify the presence of cxcl18b-defined MG transitional states, we first examined the temporal relationship of cxcl18b expression and MG proliferation after the cone ablation using in situ hybridization combined with immunostaining of either BLBP (an MG maker) or PCNA (a proliferative cell maker) (Figure 2A and Figure 2—figure supplement 1A). The result showed that as early as 24 hpi, the number of cxcl18b+ MG was rapidly peaked with no emergence of proliferative MG (11±4, n=10 in cxcl18b+ MG; mean ± SEM), and then cxcl18b+ MG continued declining in number over time and reached the lowest level since 96 hpi (1±1, n=7; mean ± SEM; Figure 2B). In contrast, the number of proliferative MG (PCNA+) peaked at 72 hpi and decreased to the lowest level at 120 hpi (9±2, n=11 in 72 hpi retina; 1±1, n=5 in 120 hpi retina; mean ± SEM; Figure 2B). Note that cxcl18b+ MG was mostly proliferative at 72 hpi (Figure 2—figure supplement 1B). 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. https://cdn.elifesciences.org/articles/106274/elife-106274-fig2-data3-v1.xlsx Figure 2—source data 4 Quantification of GS+/cxcl18b: Cre+ and GS+/cxcl18b: Cre⁻ Müller glia (MG) in the central retina region. https://cdn.elifesciences.org/articles/106274/elife-106274-fig2-data4-v1.xlsx Figure 2—source data 5 Quantification of cxcl18+ Müller glia (MG), microglia, and PCNA+ MG in the immunosuppression experiment. https://cdn.elifesciences.org/articles/106274/elife-106274-fig2-data5-v1.xlsx To further verify the temporal expression of cxcl18b in MG following the cone ablation, we created a new transgenic reporter Tg(cxcl18b: GFP) by cloning a 3k-bp-long cis-element of 5’UTR with GFP, allowing real-time monitoring of injury-induced cxcl18b expression in vivo (Figure 2C and Figure 2—figure supplement 1C and D). Combining this line with PCNA immunostaining, we confirmed the remarkable increase of cxcl18b expression (GFP+) at 48 hpi (0±1, n=4 in uninjured retinas vs 11±4, n=7 in 48 hpi; mean ± SEM; Figure 2D and E). Due to the prolonged stay of GFP protein, we could also observe that some GFP+ MG were also PCNA+ (Figure 2D). The result of proliferative MG as a subpopulation of cxcl18b+ MG led to an outstanding question as to whether cxcl18b-defined MG transitional states represented an essential route to injury-induced proliferation. To address it, we created a new transgene Tg(cxcl18b: Cre-vmhc: mCherry:: ef1α: loxP-DsRed-loxP-EGFP:: lws2: nfsb-mCherry) to perform the clonal analysis of historical cxcl18b-expressing in MG after the G/R cone ablation (details in Materials and methods; Figure 2—figure supplement 1E). After ablating the G/R cone by MTZ treatment at 6 dpf for 3 consecutive days, we found that the number of cxcl18b lineage-traced MG (marked by GFP+) and PCNA+ MG was significantly increased at 72 hpi (Figure 2F). Further analysis showed that all PCNA+ MG were cxcl18b lineage-traced MG, indicating the cxcl18b lineage-traced MG were the ones who could eventually enter into the cell cycle (13±2 cells for PCNA+ vs 13±2 cells PCNA+ and GFP+ MG; percentage of PCNA+ and cxcl18b+ vs PCNA+=93 ± 2%; n=14; p=0.19; mean ± SEM; Figure 2G). Further analysis showed that at 120 hpi, the time point that the MG proliferation has largely ceased, GFP+ MG with the lineage history of injury-induced cxcl18b expression constituted about 44% of GS+ MG at the central retina, indicating that only about half of MG could enter cxcl18b+ transitional states following the cone ablation (14±3 in GS+ and GFP+ MG; 28±4 in GS+ and GFP- MG, n=14; p<0.05; mean ± SEM; Figure 2I). Together, our clonal analysis demonstrated that proliferative MG mostly originated from cxcl18b-defined MG transitional states, and 44% central MG could become cxcl18b positive. To investigate whether cxcl18b was required for MG proliferation following G/R cone ablation, we employed CRISPR-Cas9-mediated gene disruption, using two sgRNAs targeting cxcl18b (Figure 2—figure supplement 1F and G). We found that cxcl18b knockout did not reduce MG proliferation after G/R cone ablation at 72 hpi (13±3, n=11 in WT; 11±3, n=7 in scramble sgRNA-injected; 13±3, n=7 in cxcl18b sgRNA-injected; mean ± SEM), suggesting that cxcl18b per se does not regulate MG proliferation directly (Figure 2—figure supplement 1H and I). This led us to wonder about the induction of cxcl18b-defined MG transitional states. As an inflammatory chemokine, cxcl18b serves as a reliable marker of inflammation and regulates neutrophil recruitment to injury sites (Goumenaki et al., 2024; Torraca et al., 2017). Inflammation has been previously shown to be critical for inducing regenerative responses in adult zebrafish, where it promotes reactive microglia/macrophages and MG proliferation in the retina (Iribarne and Hyde, 2022; Kyritsis et al., 2012). Notably, suppressing the immune response using dexamethasone (Dex) in zebrafish retina reduced microglial reactivation and significantly decreased the number of proliferative MG (Silva et al., 2020; Zhang et al., 2020). In our study, we identified the cxcl18b-defined transitional states as the essential routing for MG proliferation after G/R cone ablation. These results prompted us to investigate whether the inflammatory responses mediated by recruited microglia are indispensable for the formation of these cxcl18b-defined transitional states. To address this, we examined cxcl18b expression using Tg(cxcl18b: GFP) after inhibiting inflammation using Dex (Iribarne and Hyde, 2022) and observed a significant reduction in the number of cxcl18b+ MG (GFP+ cells) at 72 hpi (11±4, n=10 in Dex-treated retina vs 19±5, n=6 in DMSO treatment; and 18±3, n=14 in the 72 hpi retina; mean ± SEM) (Figure 2K). Consistent with earlier reports, we observed that Dex treatment inhibited the migration of microglia (indicated by Tg(mpeg1: GFP); 16±4, n=14 in Dex-treated retina vs 27±5, n=7 in DMSO treatment; and 28±4, n=9 in the 72 hpi retina; mean ± SEM) to the ONL and significantly reduced the number of proliferative MG (PCNA+; 8±2, n=14 in Dex-treated retina vs 17±2, n=7 in DMSO treatment; and 14±3, n=9 in the 72 hpi retina; mean ± SEM) at 72 hpi after G/R cone ablation (Figure 2L and M and Figure 2—figure supplement 1J). These findings suggest that microglia-mediated inflammation may contribute to the activation of cxcl18b-defined transitional states that precede MG proliferation, although a causal relationship remains to be established. While Dex suppressed both microglial recruitment and cxcl18b+ MG generation, its broad anti-inflammatory action precludes definitive conclusions about microglial causality. Dissecting this relationship would require concurrent ablation of microglia and cone photoreceptors using a triple-transgenic strategy, which is beyond the scope of the current study. Targeted approaches will be necessary to resolve the specific role of microglia in initiating cxcl18b expression. Previous studies have demonstrated the involvement of redox signaling in cell regeneration processes in various tissues across species (Han et al., 2014; Hunter et al., 2018; Matrone et al., 2021; Yoo et al., 2012). All this evidence led us to directly test the roles of redox genes in serving as the molecular mechanism underlying injury-induced MG proliferation. Thus, we first examined the expression levels of a comprehensive list of redox genes in cxcl18b-defined MG transitional states (Cluster 1/2/5) in our scRNA-seq data (Figure 1H) and screened the influence of 18 genes from each major category of redox signaling on injury-induced MG proliferation using CRISPR-Cas9-mediated gene disruption (Figure 4—figure supplement 1A). We focused on the NO signaling pathway, targeting three genes encoding NO synthases (Nos): neuronal Nos (nos1) and two inducible forms (nos2a and nos2b), as well as the gene encoding S-nitrosoglutathione reductase (gsnor or adh5), which modulates reactive NO signaling (Figure 4A and Figure 4—figure supplement 1B). The consequence and efficiency of gene disruption were verified by DNA sequencing (Figure 4—figure supplement 1C). Notably, the disruption of nos2b resulted in a significant reduction of PCNA+ MG at 72 hpi (6±2, n=22 in nos2b-disrupted vs 11±3, n=7 in scramble sgRNA-injected; mean ± SEM) (Figure 4B). Noted that nos gene disruption did not significantly alter microglia recruitment or G/R cone ablation at 72 hpi, suggesting that the influence of NO on injury-induced MG proliferation was not via inflammatory reactions of recruited microglia or injury degree (Figure 4—figure supplement 1D–F). Figure 4—source data 1 Quantitative analysis of PCNA+ Müller glia (MG) in the Nos metabolism gene disruption experiment after retinal injury. https://cdn.elifesciences.org/articles/106274/elife-106274-fig4-data1-v1.xlsx 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. https://cdn.elifesciences.org/articles/106274/elife-106274-fig4-data3-v1.xlsx To further examine the function of Nos2b via NO, we employed various Nos inhibitors (L-NG-nitro arginine methyl ester, L_NAME; L-NG-monomethyl arginine, L_NMMA; 1400W) and NO scavengers (carboxy-PTIO, C-PTIO) (Goldstein et al., 2003; Hong et al., 2012; Moore et al., 1990; Rees et al., 1990). We performed the intraocular injection of the drugs (PBS as control) into the zebrafish eye from 2 days before cell ablation until 72 hpi (Figure 4C). Notably, the NO scavenger C-PTIO mostly suppressed MG proliferation, indicating the involvement of NO (cell number of PCNA+ MG: 3±2, n=27 after blocking NO by C-PTIO vs 14±4, n=16 in PBS-injected retina; mean ± SEM) (Figure 4D and E). Moreover, 1400W (an inhibitor specific to inducible Nos, 7±2, n=10; mean ± SEM) and L_NAME (a broad inhibitor to all three Nos forms, 8±2, n=12; mean ± SEM) could also significantly reduce the number of proliferative MG after the ablation, whereas L_NMMA (the inhibitor specific to neuronal Nos) did not influence MG proliferation (11±3, n=12; mean ± SEM) (Figure 4E). Taken together, these results highlight the critical role of NO signaling in regulating injury-induced MG proliferation. We further generated NO pathway mutant zebrafish (nos1, nos2a, nos2b, and gsnor) to investigate the role of NO in MG proliferation following G/R cone ablation. Utilizing CRISPR/Cas9-mediated gene disruption, we successfully screened out nos1, nos2a, nos2b, and gsnor mutants, characterized by deletions of 133 bp, 13 bp, 220 bp, and 11 bp coding sequences, respectively (see Materials and methods for details, Figure 4—figure supplement 2A and B). Consistent with the gene disruption experiments described above, we observed a significant reduction in the number of proliferative (PCNA+) MG at 72 hpi following G/R cone ablation in both heterozygous and homozygous nos2b mutants (nos2b+/⁻: 3 ± 1, n = 20; nos2b⁻/⁻: 6 ± 2, n = 20) compared to WT controls (11 ± 2, n = 14; mean ± SEM). In contrast, no significant changes in MG proliferation were observed in nos1, nos2a, or gsnor mutants compared to wild type (WT) (Figure 4F–I). Interestingly, the reduction in proliferative MG was more pronounced in nos2b heterozygous mutants (nos2b+/-) than in homozygous mutants (nos2b-/-) (nos2b+/- vs nos2b-/-; p<0.001; mean ± SEM) (Figure 4J). We observed no significant difference in the loss of cone photoreceptor at 72 hpi between nos2b mutants and WT, indicating that the reduced MG proliferation observed in nos2b mutants is independent of the injury (WT: 45 ± 8 remaining cones, n = 24; nos2b+/⁻: 49 ± 12, n = 20; nos2b⁻/⁻: 46 ± 9, n = 20; mean ± SEM) (Figure 4K). This unexpected result suggests a concentration-dependent effect of NO on proliferative MG. Specifically, compared to homozygous mutants, heterozygous mutants with intermediate NO levels more effectively suppressed MG proliferation, whereas WT animals with higher NO levels promoted MG proliferation. This concentration-response pattern highlights the role of NO as a regulator, rather than a mediator, of injury-induced MG proliferation. Previous studies have shown that ascl1α and Notch signaling are essential for MG proliferation in the injured zebrafish retina (Conner et al., 2014; Wan et al., 2012). Regarding Notch signaling, a high Notch3 expression is reported to maintain MG quiescence. In response to the injury, notch3 expression is downregulated, but notch1a is necessary for the continued proliferation of the progenitors (Campbell et al., 2022). Consistently, we observed that notch3 and hey (the Notch downstream target) were highly expressed in uninjured MG clusters and became reduced from the early transitional state (Cluster 4) to the proliferative states (Clusters 3 and 6), whereas notch1a/1b and ascl1α were prominently expressed in the late stage of cxcl18b+ transitional states (Cluster 5) and the proliferative states (Cluster 3; Figures 1 and 7). Interestingly, upstream regulators of Notch signaling activation, such as fgf8a, fgf8b (Wan and Goldman, 2017), and tgfb3 (Lee et al., 2020), were predominantly expressed in Clusters 4 and 1, preceding the expression of cxcl18b (Figure 7A). These results led us to wonder whether NO regulated cxcl18b-defined transitional state MG cell-cycle re-entry via the Notch signaling pathway. Figure 7 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 To examine the influence of NO signaling blockage on Notch activity dynamics following the cone ablation, we employed a reporter line Tg(Tp1bglob: EGFP) (referred to as Tg(Tp1:EGFP)), in which EGFP is driven by the TP1 element, the direct target of the intracellular domain of Notch receptors (NICD) that is generated upon Notch activation (Parsons et al., 2009; Quillien et al., 2014). We treated fish with the NO scavenger C-PTIO and MTZ, starting at 5 dpf for 5 continuous days and at 7 dpf for 3 continuous days, respectively, followed by immunostaining for EGFP and PCNA (Figure 7B). Interestingly, Tp1: EGFP+ MG were significantly reduced at all three injury time points (cell number of EGFP+ clones: 34±3, n=9 in 24 hpi retina; 29±3, n=4 in 48 hpi retina; 33±4, n=6 in 72 hpi retina vs 42±1, n=4 in the uninjured retina; mean ± SEM), demonstrating a decrease in Notch activity following G/R cone ablation (Figure 7C). Notably, this reduction in Notch activation was further rescued by NO blocking using C-PTIO (cell number of EGFP+ clones: 42±1, n=6 in C-PTIO-treated retina at 24 hpi; 42±2, n=5 at 48 hpi; 44±2, n=7 at 72 hpi; mean ± SEM), suggesting that NO modulates Notch signaling (Figure 7C). Meanwhile, C-PTIO treatment significantly reduced the number of proliferative MG (marked by PCNA) (Figure 7D). These findings indicated that injury-induced NO suppresses Notch signaling activation, which potentially drives MG to exit quiescence and enter proliferation. Together, our results highlight that NO signaling drove the injury-induced cxcl18b-defined transitional state MG to enter proliferation, which may be mediated by Notch pathway regulation. Combined scRNA-seq analysis and clonal analysis, we identified a previously unreported transitional MG state, marked by the expression of cxcl18b, as the essential routing for MG to re-enter the cell cycle following the retinal injury (Figure 1H, Figure 2F and G). To our knowledge, it is the first transitional state verified by in vivo clonal analysis to show a faithful prediction of injury-induced MG proliferation. Notably, this cxcl18b induction in MG depends on microglial recruitment in response to cone ablation, suggesting this transitional state is an MG response to the signals derived from the inflammatory reaction (Figure 2K). The underlying mechanism of this crosstalk is crucial to be addressed. Interestingly, the cxcl18b-containing gene module was also expressed in the CMZ, a region crucial for adult retina neurogenesis for a lifetime (Figure 3A–D). However, its expression is mainly absent from the central regions of the developing retina (Figure 3F). It suggests that the cxcl18b-defined transitional state might represent a developmental state used by constitutive neuron generation programs and injury-induced neuron regeneration programs beyond embryonic development. Furthermore, the cxcl18b-defined transitional state exhibits robust redox-related characteristics, such as the expression of sod1, sod2, and catalase (Figure 4—figure supplement 1A). It led to the critical discovery of this study, which showed the essential role of nos2b in regulating injury-induced MG proliferation. Unfortunately, our preliminary effects on gene disruption using CRISPR/Cas9 in F0 founders did not observe a significant reduction in the number of proliferative MG after cxcl18b disruption (Figure 2—figure supplement 1I). However, a recent study reported the essential role of cxcl18b in heart regeneration using mutant fish, providing a mechanism of cxcl18b as innate immune signaling in injury-induced tissue regeneration (Goumenaki et al., 2024). Their results raised concern about the efficiency of cxcl18b disruption in our system. It is essential to use mutant fish to re-examine the role of cxcl18b in injury-induced MG proliferation in the future. Also, we cannot rule out the possibility that other co-factors are involved in the action of cxcl18b in MG regeneration, which is another critical issue that needs to be solved in the future. Our study, for the first time, demonstrated an essential role of NO signaling in regulating MG proliferation after the cone ablation. However, we still need to understand more about the underlying mechanism. There are two well-characterized molecular events responsible for MG proliferation following the retinal injury: a decreased Notch activity and an increased ascl1α expression (Fausett et al., 2008; Jorstad et al., 2017; Ramachandran et al., 2010). Previous studies have shown that Notch3 is responsible for this decreased Notch activity, leading to increased Ascl1a through the de-depression mechanism (Campbell et al., 2021; Sahu et al., 2021). Interestingly, unlike notch3, notch1a has been reported to stimulate MG proliferation (Campbell et al., 2021; Campbell et al., 2022). Our scRNA-seq analysis also showed that as MG progressed into the proliferative states after the cone ablation, notch3 expression gradually declined (Figure 7A). In contrast, ascl1α, notch1a, and notch1b expressions were upregulated and peaked at the proliferative states (Figure 7A). Thus, both previous studies and our current analysis support the idea that the transcriptional regulation of Notch expression accounts for the decreased Notch activity after the injury. Intriguingly, NO has been reported to activate the Notch1 signaling cascade by promoting the release and accumulation of the Notch1 intracellular domain (NICD) through nitration reactions, subsequently enhancing tumorigenesis and stem-like features in various cellular systems (Charles et al., 2010; López-Juárez et al., 2017; Villegas et al., 2018). It raises the possibility that NO signaling regulates injury-induced MG proliferation through the posttranslational modification of Notch3. Previous studies have demonstrated two significant forms of NO-mediated post-modification: S-nitrosylation on cysteines and nitration on tyrosine (Wu et al., 2014). Our preliminary analysis showed that all 11 cysteine residues within the putative γ-secretase-dependent cleavage sites are conserved between Notch1a and Notch3, while notable differences were observed at four tyrosine residues. It leads to an outstanding question of whether NO regulated injury-induced MG proliferation by decreasing Notch activity via tyrosine nitration of Notch3, which is worthwhile to elucidate. Previous studies reported that cxcl18b is a reliable inflammatory marker (Goumenaki et al., 2024; Torraca et al., 2017), and different inflammation responses modulate MG proliferation in the damaged zebrafish retina (Iribarne and Hyde, 2022). The induction of cxcl18b may represent the inflammatory responses of MG after the cone ablation, pointing out the potential link between the inflammatory response and the emergence of NO signaling in MG. Previous studies have demonstrated that iNOS is induced in various tissues by proinflammatory cytokines (Förstermann and Sessa, 2012; Pacher et al., 2007). One of the approaches to test the role of inflammatory responses is to manipulate the levels of inflammatory responses in MG to see the NO production and MG proliferative behaviors. Also, previous studies appreciate the essential role of electrical activity in tissue regeneration (Levin, 2009; Qin et al., 2023); in particular, calcium signaling has been shown to regulate various molecular pathways for liver regeneration, including the hepatocyte growth factor-Met-tyrosine kinase (HGF-Met) transduction pathway (Bedi et al., 2024) and the epidermal growth factor receptor signaling (Kimura et al., 2023). It is interesting to speculate that the abnormal electrical activity of MG in the injured retina may result in an elevated level of intracellular calcium, which activates calmodulin and induces the conformation change of NOS to NO production (Hanson et al., 2018; Jones et al., 2007). A similar mechanism has been proposed in the long-term potentiation of excitatory postsynaptic structure (Grover and Teyler, 1990; Kawamoto et al., 2012; Park, 2018), as well as in the glutamate neurotoxicity model (Ashpole et al., 2013; Ashpole et al., 2012). Thus, the production of NO derived from Nos may be the product of the interplay between the inflammatory responses and the electrical activity in MG after the retina damage.