Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing.

Neuron (2018)

2018 Dec 31

Li Q, Cheng Z, Zhou L, Darmanis S, Neff NF, Okamoto J, Gulati G, Bennett ML, Sun LO, Clarke LE, Marschallinger J, Yu G, Quake SR, Wyss-Coray T, Barres BA.
| DOI: 10.1016/j.neuron.2018.12.006

Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.

Basolateral to Central Amygdala Neural Circuits for Appetitive Behaviors

Neuron.

2017 Mar 22

Kim J, Zhang X, Muralidhar S, LeBlanc SA, Tonegawa S.
PMID: 28334609 | DOI: 10.1016/j.neuron.2017.02.034

Basolateral amygdala (BLA) principal cells are capable of driving and antagonizing behaviors of opposing valence. BLA neurons project to the central amygdala (CeA), which also participates in negative and positive behaviors. However, the CeA has primarily been studied as the site for negative behaviors, and the causal role for CeA circuits underlying appetitive behaviors is poorly understood. Here, we identify several genetically distinct populations of CeA neurons that mediate appetitive behaviors and dissect the BLA-to-CeA circuit for appetitive behaviors. Protein phosphatase 1 regulatory subunit 1B+ BLA pyramidal neurons to dopamine receptor 1+ CeA neurons define a pathway for promoting appetitive behaviors, while R-spondin 2+ BLA pyramidal neurons to dopamine receptor 2+ CeA neurons define a pathway for suppressing appetitive behaviors. These data reveal genetically defined neural circuits in the amygdala that promote and suppress appetitive behaviors analogous to the direct and indirect pathways of the basal ganglia.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential

Cell Genomics

2023 Apr 01

Carlessi, R;Denisenko, E;Boslem, E;Köhn-Gaone, J;Main, N;Abu Bakar, N;Shirolkar, G;Jones, M;Beasley, A;Poppe, D;Dwyer, B;Jackaman, C;Tjiam, M;Lister, R;Karin, M;Fallowfield, J;Kendall, T;Forbes, S;Gray, E;Olynyk, J;Yeoh, G;Forrest, A;Ramm, G;Febbraio, M;Tirnitz-Parker, J;
| DOI: 10.1016/j.xgen.2023.100301

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2

Cell host & microbe

2022 Dec 09

Barnett, KC;Xie, Y;Asakura, T;Song, D;Liang, K;Taft-Benz, SA;Guo, H;Yang, S;Okuda, K;Gilmore, RC;Loome, JF;Oguin Iii, TH;Sempowski, GD;Randell, SH;Heise, MT;Lei, YL;Boucher, RC;Ting, JP;
PMID: 36563691 | DOI: 10.1016/j.chom.2022.12.005

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes

eLife

2022 Jan 19

Yu, H;Rubinstein, M;Low, MJ;
PMID: 35044906 | DOI: 10.7554/eLife.72883

Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use single-cell RNA-sequencing (scRNA-seq) to define the transcriptomes characterizing Pomc-expressing cells in the developing hypothalamus and translating ribosome affinity purification with RNA-sequencing (TRAP-seq) to analyze the subsequent translatomes of mature POMC neurons. Our data showed that Pomc-expressing neurons give rise to multiple developmental pathways expressing different levels of Pomc and unique combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts, represents the canonical arcuate POMC neurons. Additional cell clusters expressing medium or low levels of Pomc mature into different neuronal phenotypes featured by distinct sets of transcription factors, neuropeptides, processing enzymes, cell surface, and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to mature postnatally.

A harmonized atlas of mouse spinal cord cell types and their spatial organization

Nature communications

2021 Sep 29

Russ, DE;Cross, RBP;Li, L;Koch, SC;Matson, KJE;Yadav, A;Alkaslasi, MR;Lee, DI;Le Pichon, CE;Menon, V;Levine, AJ;
PMID: 34588430 | DOI: 10.1038/s41467-021-25125-1

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

System-wide transcriptome damage and tissue identity loss in COVID-19 patients

Cell Reports Medicine

2022 Jan 01

Park, J;Foox, J;Hether, T;Danko, D;Warren, S;Kim, Y;Reeves, J;Butler, D;Mozsary, C;Rosiene, J;Shaiber, A;Afshin, E;MacKay, M;Rendeiro, A;Bram, Y;Chandar, V;Geiger, H;Craney, A;Velu, P;Melnick, A;Hajirasouliha, I;Beheshti, A;Taylor, D;Saravia-Butler, A;Singh, U;Wurtele, E;Schisler, J;Fennessey, S;Corvelo, A;Zody, M;Germer, S;Salvatore, S;Levy, S;Wu, S;Tatonetti, N;Shapira, S;Salvatore, M;Westblade, L;Cushing, M;Rennert, H;Kriegel, A;Elemento, O;Imielinski, M;Rice, C;Borczuk, A;Meydan, C;Schwartz, R;Mason, C;
| DOI: 10.1016/j.xcrm.2022.100522

The molecular mechanisms underlying the clinical manifestations of COVID-19 and what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome, remains poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match this data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue compartment-specific damage wrought by SARS-CoV-2 infection, evident as a function of varying viral loads during the clinical course of infection and tissue type specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.

Skin basal cell carcinomas assemble a pro-tumorigenic spatially organized and self-propagating Trem2+ myeloid niche

Nature communications

2023 May 10

Haensel, D;Daniel, B;Gaddam, S;Pan, C;Fabo, T;Bjelajac, J;Jussila, AR;Gonzalez, F;Li, NY;Chen, Y;Hou, J;Patel, T;Aasi, S;Satpathy, AT;Oro, AE;
PMID: 37164949 | DOI: 10.1038/s41467-023-37993-w

Cancer immunotherapies have revolutionized treatment but have shown limited success as single-agent therapies highlighting the need to understand the origin, assembly, and dynamics of heterogeneous tumor immune niches. Here, we use single-cell and imaging-based spatial analysis to elucidate three microenvironmental neighborhoods surrounding the heterogeneous basal cell carcinoma tumor epithelia. Within the highly proliferative neighborhood, we find that TREM2+ skin cancer-associated macrophages (SCAMs) support the proliferation of a distinct tumor epithelial population through an immunosuppression-independent manner via oncostatin-M/JAK-STAT3 signaling. SCAMs represent a unique tumor-specific TREM2+ population defined by VCAM1 surface expression that is not found in normal homeostatic skin or during wound healing. Furthermore, SCAMs actively proliferate and self-propagate through multiple serial tumor passages, indicating long-term potential. The tumor rapidly drives SCAM differentiation, with intratumoral injections sufficient to instruct naive bone marrow-derived monocytes to polarize within days. This work provides mechanistic insights into direct tumor-immune niche dynamics independent of immunosuppression, providing the basis for potential combination tumor therapies.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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