Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence
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Evaluation Summary:
This work convincingly provides important novel mechanisms on how microglial states are shaped during retinal development, an important question that should be of interest for a broad readership.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and dependence on Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drive multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia toward distinct remodeling states and through Axl, alter microglial dependence on survival pathway, CSF1R.
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Author Response:
We thank the reviewers for their thoughtful evaluation of the manuscript and insightful comments. One reviewer commented on the lack of biological replicates for our scRNAseq. To mitigate batch effects, we sequenced a single technical replicate for each condition, but each consisted of several biological specimens (11-13 animals each) from multiple litters, totaling 49 animals. Pooling this large number of retinas was necessary for obtaining enough cells for analysis, and consequently, we have many biological samples represented. In addition, the proportions of microglia expressing specific markers in our scRNAseq match our published quantification by in situ hybridization (Anderson et al. Cell Reports 2019), giving us confidence that the proportions determined here are representative. The reviewer also asked whether …
Author Response:
We thank the reviewers for their thoughtful evaluation of the manuscript and insightful comments. One reviewer commented on the lack of biological replicates for our scRNAseq. To mitigate batch effects, we sequenced a single technical replicate for each condition, but each consisted of several biological specimens (11-13 animals each) from multiple litters, totaling 49 animals. Pooling this large number of retinas was necessary for obtaining enough cells for analysis, and consequently, we have many biological samples represented. In addition, the proportions of microglia expressing specific markers in our scRNAseq match our published quantification by in situ hybridization (Anderson et al. Cell Reports 2019), giving us confidence that the proportions determined here are representative. The reviewer also asked whether the clusters with remodeling signatures are actually remodeling. We agree with the reviewer that we cannot infer function using transcriptomic data alone. However, our scRNAseq of microglia illustrates several clusters with high expression of lysosomal and lipid metabolism genes which are diminished or absent in retinas lacking developmental apoptosis (Bax KO), consistent with remodeling activity. A complete in vivo functional analysis of each remodeling cluster is beyond the scope of this manuscript, but we are currently probing this further for another publication.
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Evaluation Summary:
This work convincingly provides important novel mechanisms on how microglial states are shaped during retinal development, an important question that should be of interest for a broad readership.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this interesting and clearly written paper, which continues a line of work that originated with two 2019 papers from the same authors, Anderson et al. investigate the transcriptional diversity of microglia in developing mouse retina and how this diversity is impacted by phagocytosis of apoptotic neurons. Using single-cell RNA-seq they find substantial diversity among retinal microglia at P6/7 that echoes the diversity of microglia in early postnatal brain. Unlike in the brain, few P6-P7 retinal microglia are in the well-characterized homeostatic resting state. Instead, based on their gene profiles, the authors surmise that most retinal microglia are in a phagocytic (or "remodeling") state. Striking evidence for this hypothesis was provided by scRNA-seq experiments in which neuronal apoptosis was …
Reviewer #1 (Public Review):
In this interesting and clearly written paper, which continues a line of work that originated with two 2019 papers from the same authors, Anderson et al. investigate the transcriptional diversity of microglia in developing mouse retina and how this diversity is impacted by phagocytosis of apoptotic neurons. Using single-cell RNA-seq they find substantial diversity among retinal microglia at P6/7 that echoes the diversity of microglia in early postnatal brain. Unlike in the brain, few P6-P7 retinal microglia are in the well-characterized homeostatic resting state. Instead, based on their gene profiles, the authors surmise that most retinal microglia are in a phagocytic (or "remodeling") state. Striking evidence for this hypothesis was provided by scRNA-seq experiments in which neuronal apoptosis was eliminated (using Bax mutants). When there are no apoptotic neurons to engulf, the authors find that most microglia become homeostatic, with matching reductions in remodeling cell clusters. Another interesting set of experiments investigates microglial survival factors and the role of TAM receptors Mer and Axl in phagocytosis/survival. Surprisingly, unlike previous reports in brain, Mer and Axl have distinct roles in retina. Mer is needed for clearance of apoptotic neurons, as expected, but Axl is important for microglial survival, especially in the context of Csf1R blockade.
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Reviewer #2 (Public Review):
In the article titled "Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence" the authors describe the heterogeneity of the microglial population in the early postnatal retina. In WT mice, the authors find both "homeostatic" microglia (expressing Tmem119 and P2RY12) and several clusters of "remodeling" microglia (expressing lysosomal and phagocytic components). In a Bax KO mouse lacking the significant apoptosis that usually occurs in the early postnatal retina, they find a decrease in the remodeling clusters of microglia, concluding that the lack of apoptosis in this mouse prevents the shift in microglial phenotype from homeostatic to remodeling. Several of these "remodeling" clusters in WT mice are relatively enriched after short term PLX3397 treatment, a CSF1R …
Reviewer #2 (Public Review):
In the article titled "Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence" the authors describe the heterogeneity of the microglial population in the early postnatal retina. In WT mice, the authors find both "homeostatic" microglia (expressing Tmem119 and P2RY12) and several clusters of "remodeling" microglia (expressing lysosomal and phagocytic components). In a Bax KO mouse lacking the significant apoptosis that usually occurs in the early postnatal retina, they find a decrease in the remodeling clusters of microglia, concluding that the lack of apoptosis in this mouse prevents the shift in microglial phenotype from homeostatic to remodeling. Several of these "remodeling" clusters in WT mice are relatively enriched after short term PLX3397 treatment, a CSF1R inhibitor commonly used to deplete microglia in mouse.
This paper provides interesting new phenotyping of retinal microglia and how they change with Bax KO and PLX treatment and has potential to be an impactful contribution. In particular, the idea that microglia engaged in tissue remodeling might be less CSF1R dependent could guide the development of new treatments and improve our fundamental understanding of eye development. At present, the central claim and primary new finding compared to a prior study from this group (Anderson et al 2019, Cell Reports), is that the identified remodeling clusters are not only CSF1R independent, but more specifically that expression of remodeling genes confers this survival advantage. On initial submission, this very interesting claim is partially supported by the data presented, but this support is greatly limited by technical issues (a lack of biological replicates for single cell data), and a lack of assessment of whether clusters inferred to be performing remodeling functions are actually doing so.
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Reviewer #3 (Public Review):
The strength of the manuscript lies in its thorough and comprehensive characterization of multiple microglia states in the developing postnatal mouse retina. By the mean of scRNAseq analysis, the authors addressed the question of which environmental cues drive such heterogeneity.
This work allowed the identification of 11 microglia states that coexist in wild type postnatal retina, ranging from a so-called homeostatic state to some remodeling states, with for instance a disease-associated signature. The proportion of the different microglia states is context-dependent and the authors demonstrated that the spectrum of homeostatic to more remodeling clusters is driven predominately by neuronal apoptosis (resulting from waves of neuronal cell death during the postnatal period in the retina). On the other hand, …
Reviewer #3 (Public Review):
The strength of the manuscript lies in its thorough and comprehensive characterization of multiple microglia states in the developing postnatal mouse retina. By the mean of scRNAseq analysis, the authors addressed the question of which environmental cues drive such heterogeneity.
This work allowed the identification of 11 microglia states that coexist in wild type postnatal retina, ranging from a so-called homeostatic state to some remodeling states, with for instance a disease-associated signature. The proportion of the different microglia states is context-dependent and the authors demonstrated that the spectrum of homeostatic to more remodeling clusters is driven predominately by neuronal apoptosis (resulting from waves of neuronal cell death during the postnatal period in the retina). On the other hand, microglia-expressed recognition receptors, Mer or Axl, required for clearance of apoptotic cells and survival in the absence of CSF1R signaling, respectively, do not mediate changes in microglia remodeling gene expression.
The manuscript is extremely clear and the data are thoroughly described. They perfectly support the main conclusions. Although the finding that developmental apoptosis impact retinal microglia gene signature is not novel (a manuscript from the authors and based on bulk RNAseq analysis was published in Cell Reports in 2019), the comprehensive scRNA-seq clustering analysis is an important addition, providing a valuable resource for the field.
As true for most of scRNAseq analysis, the authors deal with lists of many markers and names of ligands/receptors, so a good knowledge in the field is advised for an easy reading of the manuscript.
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