Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain

Marsh, Samuel E.; Walker, Alec J.; Kamath, Tushar; Dissing-Olesen, Lasse; Hammond, Timothy R.; Soysa, T. Yvanka de; Young, Adam M. H.; Murphy, Sarah; Abdulraouf, Abdulraouf; Nadaf, Naeem; Dufort, Connor; Walker, Alicia C.; Lucca, Liliana E.; Kozareva, Velina; Vanderburg, Charles R.; Hong, Soyon; Bulstrode, Harry; Hutchinson, Peter J.; Gaffney, Daniel J; Hafler, David A.; Franklin, Robin J.M.; Macosko, Evan Z.; Stevens, Beth

doi:10.1038/s41593-022-01022-8

Cited by 224 publications

(223 citation statements)

References 90 publications

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“…Tmem119 ; Figure 1 ) and so we termed it Hom/chemokine cluster. Ccl3 and Ccl4 are among a set of genes that can be induced by dissociation ( Marsh et al, 2020 ) so we confirmed that retinal microglia express Ccl3 and Ccl4 in vivo by in situ hybridization ( Figure 1—figure supplement 5 and data not shown). Cluster 3 was marked by Npl , found in lipid-droplet microglia ( Marschallinger et al, 2020 ), and Apoc1 , a lipoprotein high in multiple sclerosis-associated human microglia ( Masuda et al, 2019 ).…”

Section: Resultssupporting

confidence: 69%

“…Future studies could target these specific microglia subsets and try to link them to other important remodeling functions in the developing retina. Some clusters did have higher expression of ex vivo activation genes ( Marsh et al, 2020 ) but the impact on developmental datasets has not been directly tested. exAM genes did not drive the formation of a single cluster and some genes were differentially expressed between samples, arguing against solely a technical effect.…”

Section: Discussionmentioning

confidence: 99%

“…For comparison to interferon-responsive microglia, we used cluster eight markers from Table 5 of Dorman et al, 2022 . For exAM activation genes, we used 27 genes from Table 4 of Marsh et al, 2020 .…”

Section: Methodsmentioning

confidence: 99%

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Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence

Anderson

Roberts

Ghena

et al. 2022

eLife

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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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Section: Resultssupporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

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Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence

Anderson

Roberts

Ghena

et al. 2022

eLife

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“…Another recent study also uncovered that glial cell types respond to enzymatic dissociation during single-cell and single-nucleus library preparation and confound the transcriptomic profile 46 . Here, we show that all glial cell types are also contaminated with neuronal ambient RNA transcripts.…”

Section: Discussionmentioning

confidence: 99%

Ambient RNA analysis reveals misinterpreted and masked cell types in brain single-nuclei datasets

Caglayan

Liu

Konopka

2022

Preprint

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Ambient RNA contamination in single-cell RNA sequencing (RNA-seq) is a significant problem, but its consequences are poorly understood. Here, we show that ambient RNAs in brain single-nuclei RNA-seq can have a nuclear or extra-nuclear origin with distinct gene set signatures. Both ambient RNA signatures are predominantly neuronal and we find that some previously annotated neuronal cell types are distinguished by ambient RNA. Strikingly, we also detect pervasive neuronal ambient RNA contamination in all glial cell types unless glia and neurons are physically separated prior to sequencing. We demonstrate that this contamination can be removed in silico. We also show that previous annotations of immature oligodendrocytes are likely glial cells contaminated with ambient RNAs. After ambient RNA removal, we detect extremely rare, committed oligodendrocyte progenitor cells, which were infrequently annotated in previous adult human brain datasets. Together, these results provide an in-depth analysis of ambient RNA contamination in brain single-cell datasets.

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“…Finally, as is the current standard, we have used enzymatic digestion and cell sorting to isolate EGCs from the LMMP layer. Such techniques can be confounded by artifactual gene signatures in diverse tissues, including brain microglia (Adam et al, 2017; Marsh et al, 2022). Multiome sequencing including ATAC can mitigate this problem, as ATAC signal delineates cell identity more robustly than RNA (Corces et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Single-Cell Multiome Sequencing Clarifies Enteric Glial Cell Diversity and Identifies an Intraganglionic Population Poised for Neurogenesis

Guyer

Bhave

Stavely

et al. 2021

Preprint

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Traditional models posit that enteric neurons and glial cells represent distinct terminal lineages derived from a common neural crest precursor. This model, however, does not explain the neurogenic capability of murine postnatal enteric glial cells. To characterize the full diversity of myenteric glial cells and identify a basis for the glial-to-neuronal transition, which we demonstrate using a two-marker system, we applied single-cell RNA sequencing and single-nucleus ATAC sequencing to generate a multiomic atlas of Plp1-expressing glial cells from the small intestine of adolescent mice. We identify nine transcriptionally distinct subpopulations of enteric glial cells, including cells expressing both canonical neural stem cell genes and enteric neuronal transcriptional factors. We refer to these Plp1-positive cells with neural stem cell features as glial neuroblasts. Surprisingly, most glial cells maintain open chromatin at neuronal-associated loci, suggesting enteric glia are primed for neuronal transition. Comparison with the developing embryonic enteric nervous system shows postnatal glial cells maintain a transcriptional program closely matching embryonic neuronal progenitors. Transcription factor motif enrichment analysis and regulon analysis implicate AP-1 transcription factors in maintaining the glial neuroblast gene program. Three-dimensional cultures of postnatal enteric nervous system cells, which are enriched for glial neuroblasts and provide a niche for neuronal development, recapitulate the transcriptional changes seen during embryonic enteric neurogenesis. snATAC analysis shows chromatin closing consistent with terminal differentiation as glial cells become neurons in three-dimensional culture. In conclusion, postnatal myenteric glial cells include a neuroblast population and maintain a chromatin structure primed for neuronal fate acquisition.

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Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain

Cited by 224 publications

References 90 publications

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence

Ambient RNA analysis reveals misinterpreted and masked cell types in brain single-nuclei datasets

Single-Cell Multiome Sequencing Clarifies Enteric Glial Cell Diversity and Identifies an Intraganglionic Population Poised for Neurogenesis

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