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

Reviewer #1 (Public review):

Summary:

This study presents a valuable contribution of NO signaling in zebrafish retinal regeneration in larval animals. The data on NO signaling are solid; however, the link to cxcl118b is inadequate. There are significant concerns that the RNA-seq studies largely repeat the work of a previous study done in adult animals, which is a more relevant biological variable for translational insights.

Strengths:

New data on NO signaling are valuable to the field, but may be limited to larval "regeneration".

Weaknesses:

(1) The authors state that more is known about glial reactivation than cell-cycle re-entry. They are confusing many points here. More gene networks that require cell-cycle re-entry are known. Some of the genes listed for "reactivation" are, in fact, required for cell cycle re-entry/proliferation. And the authors confuse gliosis vs glial reactivation.

(2) A major weakness of the approach is testing cone ablation and regeneration in early larval animals. For example, cones are ablated starting the day that they are born. MG that are responding are also very young, less than 48 hrs old. It is also unclear whether the immune response of microglia is a mature response. All of these assays would be of higher significance if they were performed in the context of a mature, fully differentiated, adult retina. All analysis in the paper is negatively affected by this biological variable.

(3) Related to the above point, the clonal analysis of cxcl18b+ MG is complicated by the fact that new MG are still being born in the CMZ (as are new cones for that matter).

(4) A near identical study was already done by Hoang et al., 2020, in adult zebrafish, a more relevant biological timepoint. Did the authors check this published RNA-seq database for their gene(s) of interest?

(5) KD of cxcl18b did not affect MG proliferation or any other defined outcome. And yet the authors continually state such phrases as "microglia-mediated inflammation is critical for activating the cxcl18b-defined transitional states that drive MG proliferation." This is false. Cxcl18b does not drive MG proliferation at all.

(6) A technical concern is that intravitreal injections are not routinely performed in larval fish.

Reviewer #2 (Public review):

Summary:

In this manuscript, Ye et al. examine the sequence of events that occur in the damaged zebrafish Muller glia (MG) in states between quiescence and the onset of proliferation. Using an inducible metronidazole (MTZ) and nitroreductase system to ablate red/green cones in larval zebrafish, they identify a novel transitional MG state that is characterized by the expression of cxcl18b. Using trajectory analysis from single-cell RNA-seq datasets, they find that cxcl18b is expressed before MG expression PCNA and becomes proliferative. They find that cxcl18b expression peaks in MG at approximately 24 hours post injury (hpi) and rapidly declines as MG proliferate following injury. In a most interesting finding, the authors find a link between nos2b-dependent nitric oxide signaling and cxcl18b-mediated proliferation. Mutagenesis of nos2b decreases MG proliferation. The mechanism linking NO signaling to proliferation was suggested to function via notch signaling as pharmacological inhibition of nitric oxidate signaling resulted in elevated Notch activity, thus preventing MG proliferation. The authors suggest a model whereby cxcl18b induces autocrine NO signaling in MG to reduce the activity of Notch3, thereby promoting MG proliferation. While this model is appealing, there are several limitations and inconsistencies within the data that raise concerns. Several conclusions regarding the role of nos2b rely on low-quality in situ hybridization data and RT-PCR results that are inconsistent with some single-cell RNA-seq data also provided. The temporal sequence of events lacks adequate rigor, as many conclusions are based on transgene expression in the Tg(cxcl18b:GFP) lines, where persistence of the GFP fluorescence may not reflect endogenous cxcl18b. Are cognate cxcl18b receptors found on MG to support an autocrine signaling pathway?

Strengths:

The authors utilize a number of sophisticated transgenic approaches and generate novel lines that will have value to the field. The identification of a novel cxcl18b transition state is exciting, and the putative link between NO signaling and Notch activity would provide new insight into the drivers of Muller glia proliferation.

Weaknesses:

While this model is appealing, there are several limitations and inconsistencies within the data that raise concerns. Several conclusions regarding the role of nos2b rely on low-quality in situ hybridization data and RT-PCR results that are inconsistent with some single-cell RNA-seq data also provided. The temporal sequence of events lacks adequate rigor, as many conclusions are based on transgene expression in the Tg(cxcl18b:GFP) lines, where persistence of the GFP fluorescence may not reflect endogenous cxcl18b. Are cognate cxcl18b receptors found on MG to support an autocrine signaling pathway? The images are generally well organized, although a number of typographical errors exist, and some sentences and phrases appear confusing. Additional proof-reading is strongly recommended.