Developmental Synchrony of Retinal Waves, Apoptosis, and Angiogenesis in Postnatal Retina

Reviewer #1 (Public review):

Summary:

This study presents a potentially important integrative model linking spontaneous retinal waves, apoptosis, microglial activity, and vascular development during postnatal retinal maturation. Its significance lies in proposing a mechanistic framework that could reshape understanding of how neural activity and tissue remodeling are coordinated in the developing central nervous system. The evidence is strengthened by the use of multiple complementary techniques, including Ca++ imaging, high-throughput electrophysiology, transcriptomics, histology, and pharmacology.

Strengths:

(1) Multimodal Validation: The authors correlate large-scale functional imaging (calcium imaging and MEA) with high-resolution structural and molecular data (scRNA-seq and IHC), providing strong topographical evidence for the "centrifugal expansion" pattern.

(2) The primary significance lies in identifying apoptotic Retinal Ganglion Cells (RGCs) as the physiological "pacemakers" for stage II retinal waves. By linking programmed cell death directly to neural activity and subsequent angiogenesis, the authors propose a self-regulating developmental loop.

Weaknesses:

(1) While the PANX1 pharmacological data provide compelling functional support, extending these conclusions to the broader CNS may be premature. Additional direct mechanistic validation would further strengthen the claim of causality.

(2) While the manuscript beautifully illustrates the co-occurrence of events during retinal development, strengthening the distinction between correlation and direct causation would enhance the impact of the findings.

Reviewer #2 (Public review):

Summary:

Savage et al. investigate the synchronization of retinal Ca2+ waves with developmental cell death, microglia activation, and vascular outgrowth. These developmental processes occur through a mechanism where apoptotic cells release ATP through Panx-1 channels to stimulate both Ca2+ retinal waves and microglia activation. Using scRNAseq, the authors classify autofluorescence cell clusters (ACCs) at the leading edge of vasculature outgrowth as Hmox-1+ microglia. From here, they show microglia engulfment of apoptotic RGCs, and the potential release of ATP may contribute to Ca2+ wave generation. The authors demonstrate these mechanisms through the use of two pharmacological agents to either block the ATP release from Panx-1 or block receptor binding to ATP. Furthermore, while previous studies have described the site of initiation of retinal Ca2+ waves as random, this study shows that the initiation of Ca2+ waves is biased to the leading edge of vascular growth in the developing retina. To do this, the authors use a combination of wide-field Ca2+ imaging and multi-electrode arrays to pinpoint the sites of Ca2+ wave initiation in the developing retina.

Strengths:

The authors use several techniques to interrogate these mechanisms, including single-cell RNAseq, wide-field Ca2+ imaging, and multi-electrode arrays. With these experiments, this manuscript proposes several novel ideas, such as ATP as the Ca2+ wave-initiating cue, and the localization of the Ca2+ wave initiation to the leading edge of vascular growth.

Weaknesses:

The main weakness of the manuscript is the overreliance on only two pharmacological agents to test the central hypotheses. These conclusions would be strengthened if, in addition to their pharmacological manipulations, they used genetic knockout models to perturb programmed cell death or ATP release (i.e., BAX-KO, Panx-1 KO).

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study presents a potentially important integrative model linking spontaneous retinal waves, apoptosis, microglial activity, and vascular development during postnatal retinal maturation. Its significance lies in proposing a mechanistic framework that could reshape understanding of how neural activity and tissue remodeling are coordinated in the developing central nervous system. The evidence is strengthened by the use of multiple complementary techniques, including Ca++ imaging, high-throughput electrophysiology, transcriptomics, histology, and pharmacology.

Strengths:

(1) Multimodal Validation: The authors correlate large-scale functional imaging (calcium imaging and MEA) with high-resolution structural and molecular data (scRNA-seq and IHC), providing strong topographical evidence for the "centrifugal expansion" pattern.

(2) The primary significance lies in identifying apoptotic Retinal Ganglion Cells (RGCs) as the physiological "pacemakers" for stage II retinal waves. By linking programmed cell death directly to neural activity and subsequent angiogenesis, the authors propose a self-regulating developmental loop.

We thank the reviewer for their nice summary and for highlighting the strengths of this work.

Weaknesses:

(1) While the PANX1 pharmacological data provide compelling functional support, extending these conclusions to the broader CNS may be premature. Additional direct mechanistic validation would further strengthen the claim of causality.

We agree with the reviewer that the conclusions would be greatly solidified with more direct mechanistic validation. However, we are unable to conduct more experimentation as the grant is finished and the Sernagor lab is in the process of being shutdown, after the unexpected passing of the PI.

In order to make clearer that this mechanism was found in retinal tissue, not CNS, we have moved any mention of the implications of our work to a broader CNS mechanism to the discussion section. We will add text into the discussion highlighting the need for more mechanistic investigation to uncover the full extent of the developmental processes described herein.

(2) While the manuscript beautifully illustrates the co-occurrence of events during retinal development, strengthening the distinction between correlation and direct causation would enhance the impact of the findings.

We have been clear to only present our findings as correlational as we were unable to fully explore the causational nature within the mechanisms presented. In the discussion, we have used published evidence and experimental papers to bolster our understanding of the causal aspects of this research. We will also include sections of text to address what experimentation is required to examine the causal interactions more directly.

Reviewer #2 (Public review):

Summary:

Savage et al. investigate the synchronization of retinal Ca2+ waves with developmental cell death, microglia activation, and vascular outgrowth. These developmental processes occur through a mechanism where apoptotic cells release ATP through Panx-1 channels to stimulate both Ca2+ retinal waves and microglia activation. Using scRNAseq, the authors classify autofluorescence cell clusters (ACCs) at the leading edge of vasculature outgrowth as Hmox-1+ microglia. From here, they show microglia engulfment of apoptotic RGCs, and the potential release of ATP may contribute to Ca2+ wave generation. The authors demonstrate these mechanisms through the use of two pharmacological agents to either block the ATP release from Panx-1 or block receptor binding to ATP. Furthermore, while previous studies have described the site of initiation of retinal Ca2+ waves as random, this study shows that the initiation of Ca2+ waves is biased to the leading edge of vascular growth in the developing retina. To do this, the authors use a combination of wide-field Ca2+ imaging and multi-electrode arrays to pinpoint the sites of Ca2+ wave initiation in the developing retina.

Strengths:

The authors use several techniques to interrogate these mechanisms, including single-cell RNAseq, wide-field Ca2+ imaging, and multi-electrode arrays. With these experiments, this manuscript proposes several novel ideas, such as ATP as the Ca2+ wave-initiating cue, and the localization of the Ca2+ wave initiation to the leading edge of vascular growth.

We thank the reviewer for their nice summary and for highlighting the strengths of this work.

Weaknesses:

The main weakness of the manuscript is the overreliance on only two pharmacological agents to test the central hypotheses. These conclusions would be strengthened if, in addition to their pharmacological manipulations, they used genetic knockout models to perturb programmed cell death or ATP release (i.e., BAX-KO, Panx-1 KO).

We thank the reviewer for their insightful suggestions for further experimentation to bolster the research. Initially, we utilised pharmacological interventions as they provided acute and quick answering of the research question. At the outset of the research, we were not certain that purinergic release through PANX-1 channels was the mediator for the developmental mechanisms described. We tested a wide variety of specific agonists and blockers before seeing any profound effects on wave generation. These agonists and antagonists have been used before and are proven to deliver reliable results. In addition, since the ACCs had never been reported before we were unsure if a knockout animal would display the same anatomical phenotype. Furthermore, it is known that knockout mouse lines, especially connexin and hemichannel pores, do not lose function but rather have other isoforms or compensation mechanisms which can substitute the original function. For the retina, for example, it was shown that Cx36 can functionally replace Cx45 after Cx45 KO (Frank et al, 2010).

We agree that while direct mechanistic validation would significantly reinforce the arguments, we are limited in conducting further experiments since the grant has been completed and the Sernagor lab is in the process of shutting down following her passing.

In order to address the omission of mechanistic validation in the paper we will add text into the discussion highlighting the need deeper investigation in the causality of the developmental processes described herein.

M. Frank et al., Neuronal connexin-36 can functionally replace connexin-45 in mouse retina but not in the developing heart, J. Cell Sci. 123, 3605 (2010).