Single-cell RNA transcriptome landscape of hepatocytes and non-parenchymal cells in healthy and NAFLD mouse liver

iScience

2021 Nov 01

Su, Q;Kim, S;Adewale, F;Zhou, Y;Aldler, C;Ni, M;Wei, Y;Burczynski, M;Atwal, G;Sleeman, M;Murphy, A;Xin, Y;Cheng, X;
| DOI: 10.1016/j.isci.2021.103233

Nonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers.

Delineation of an insula-BNST circuit engaged by struggling behavior that regulates avoidance in mice

Nature communications

2021 Jun 11

Luchsinger, JR;Fetterly, TL;Williford, KM;Salimando, GJ;Doyle, MA;Maldonado, J;Simerly, RB;Winder, DG;Centanni, SW;
PMID: 34117229 | DOI: 10.1038/s41467-021-23674-z

Active responses to stressors involve motor planning, execution, and feedback. Here we identify an insular cortex to BNST (insula→BNST) circuit recruited during restraint stress-induced active struggling that modulates affective behavior. We demonstrate that activity in this circuit tightly follows struggling behavioral events and that the size of the fluorescent sensor transient reports the duration of the struggle event, an effect that fades with repeated exposure to the homotypic stressor. Struggle events are associated with enhanced glutamatergic- and decreased GABAergic signaling in the insular cortex, indicating the involvement of a larger circuit. We delineate the afferent network for this pathway, identifying substantial input from motor- and premotor cortex, somatosensory cortex, and the amygdala. To begin to dissect these incoming signals, we examine the motor cortex input, and show that the cells projecting from motor regions to insular cortex are engaged shortly before struggle event onset. This study thus demonstrates a role for the insula→BNST pathway in monitoring struggling activity and regulating affective behavior.

Cell-type-specific interrogation of CeA Drd2 neurons to identify targets for pharmacological modulation of fear extinction

Transl Psychiatry

2018 Aug 22

McCullough KM, Daskalakis NP, Gafford G, Morrison FG, Ressler KJ.
PMID: 30135420 | DOI: 10.1038/s41398-018-0190-y

Behavioral and molecular characterization of cell-type-specific populations governing fear learning and behavior is a promising avenue for the rational identification of potential therapeutics for fear-related disorders. Examining cell-type-specific changes in neuronal translation following fear learning allows for targeted pharmacological intervention during fear extinction learning, mirroring possible treatment strategies in humans. Here we identify the central amygdala (CeA) Drd2-expressing population as a novel fear-supporting neuronal population that is molecularly distinct from other, previously identified, fear-supporting CeA populations. Sequencing of actively translating transcripts of Drd2 neurons using translating ribosome affinity purification (TRAP) technology identifies mRNAs that are differentially regulated following fear learning. Differentially expressed transcripts with potentially targetable gene products include Npy5r, Rxrg, Adora2a, Sst5r, Fgf3, Erbb4, Fkbp14, Dlk1, and Ssh3. Direct pharmacological manipulation of NPY5R, RXR, and ADORA2A confirms the importance of this cellpopulation and these cell-type-specific receptors in fear behavior. Furthermore, these findings validate the use of functionally identified specific cell populations to predict novel pharmacological targets for the modulation of emotional learning.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

Immunity.

2018 Nov 13

Girbl T, Lenn T, Perez L, Rolas L, Barkaway A, Thiriot A, del Fresno C, Lynam E, Hub E, Thelen M, Graham G, Alon R, Sancho D, von Andrian UH, Voisin MB, Rot A, Nourshargh S.
PMID: 30446388 | DOI: 10.1016/j.immuni.2018.09.018

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

Cell reports methods

2022 Aug 22

Agnew-Svoboda, W;Ubina, T;Figueroa, Z;Wong, YC;Vizcarra, EA;Roebini, B;Wilson, EH;Fiacco, TA;Riccomagno, MM;
PMID: 36046623 | DOI: 10.1016/j.crmeth.2022.100276

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.

Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier

Neuron

2022 Mar 10

Ayloo, S;Lazo, CG;Sun, S;Zhang, W;Cui, B;Gu, C;
PMID: 35294899 | DOI: 10.1016/j.neuron.2022.02.017

Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surfaces of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, α5, in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding, as well as endothelial-specific deletion of integrin α5, causes barrier leakage in mice. Furthermore, vitronectin-integrin α5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.

The retinal ipRGC-preoptic circuit mediates the acute effect of light on sleep

Nature communications

2021 Aug 25

Zhang, Z;Beier, C;Weil, T;Hattar, S;
PMID: 34433830 | DOI: 10.1038/s41467-021-25378-w

Light regulates daily sleep rhythms by a neural circuit that connects intrinsically photosensitive retinal ganglion cells (ipRGCs) to the circadian pacemaker, the suprachiasmatic nucleus. Light, however, also acutely affects sleep in a circadian-independent manner. The neural circuits involving the acute effect of light on sleep remain unknown. Here we uncovered a neural circuit that drives this acute light response, independent of the suprachiasmatic nucleus, but still through ipRGCs. We show that ipRGCs substantially innervate the preoptic area (POA) to mediate the acute light effect on sleep in mice. Consistently, activation of either the POA projecting ipRGCs or the light-responsive POA neurons increased non-rapid eye movement (NREM) sleep without influencing REM sleep. In addition, inhibition of the light-responsive POA neurons blocked the acute light effects on NREM sleep. The predominant light-responsive POA neurons that receive ipRGC input belong to the corticotropin-releasing hormone subpopulation. Remarkably, the light-responsive POA neurons are inhibitory and project to well-known wakefulness-promoting brain regions, such as the tuberomammillary nucleus and the lateral hypothalamus. Therefore, activation of the ipRGC-POA circuit inhibits arousal brain regions to drive light-induced NREM sleep. Our findings reveal a functional retina-brain circuit that is both necessary and sufficient for the acute effect of light on sleep.

Coordination of endothelial cell positioning and fate specification by the epicardium

Nature communications

2021 Jul 06

Quijada, P;Trembley, MA;Misra, A;Myers, JA;Baker, CD;Pérez-Hernández, M;Myers, JR;Dirkx, RA;Cohen, ED;Delmar, M;Ashton, JM;Small, EM;
PMID: 34230480 | DOI: 10.1038/s41467-021-24414-z

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

CRH Neurons in the Lateral Hypothalamic Area Regulate Food-Cue-Induced Appetite

Available at SSRN

2023 May 19

Li, S;Cao, J;Tan, K;Fan, L;Wang, Y;Shen, Z;Li, S;Wu, C;Zhou, H;Xu, H;
| DOI: 10.2139/ssrn.4451670

Food cues elicit the body’s responses and subsequent food consumption. The magnitude of the response to food cues is a crucial risk factor for obesity. However, the underlying neural mechanism of how the cues of edible food promote feeding remains unclear. Here we demonstrated that the peptidergic CRH neurons in the lateral hypothalamic area are the missing link that connects food cues to food consumption. We first established the activation of those neurons triggered by food cues with multiple assays. Manipulations using optogenetic and chemogenetic assays revealed that the activation increases appetite and promotes feeding, while inhibition decreases them. Finally, we identified the downstream targets of those neurons in the ventral tegmental area, and the locus coeruleus mediated the effect. Our research sheds light on the neural mechanism of how food cues increase appetite and promote feeding behavior.

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.

Molecular Analysis of HPV-independent Primary Endometrial Squamous Cell Carcinoma Reveals TP53 and CDKN2A comutations: A Clinicopathologic Analysis With Re-evaluation of Diagnostic Criteria

The American journal of surgical pathology

2022 Sep 05

Hopkins, MR;Palsgrove, DN;Ronnett, BM;Vang, R;Lin, J;Murdock, TA;
PMID: 36069815 | DOI: 10.1097/PAS.0000000000001970

Human papillomavirus (HPV)-independent primary endometrial squamous cell carcinoma (PESCC) is a rare but aggressive subtype of endometrial carcinoma for which little is known about the genomic characteristics. Traditional criteria have restricted the diagnosis of PESCC to cases without any cervical involvement. However, given that modern ancillary techniques can detect HPV and characteristic genetic alterations that should identify the more common mimics in the differential diagnosis, including endometrial endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma, those criteria may benefit from revision. To further characterize PESCC, we identified 5 cases of pure squamous cell carcinoma dominantly involving the endometrium that had the potential to be PESCC: 1 case involving only the endometrium and 4 cases with some involvement of the cervix. Clinicopathologic features were assessed and immunohistochemical analysis (p16, estrogen receptor, progesterone receptor, and p53), HPV RNA in situ hybridization (high-risk and low-risk cocktails and targeted probes for 16 and 18), and molecular studies were performed. All tumors showed aberrant/mutation-type p53 expression, were negative for estrogen receptor, progesterone receptor, and p16, and had no detectable HPV. Per whole-exome sequencing, 4 of the 5 tumors demonstrated comutations in TP53 and CDKN2A (p16). Four patients died of disease within 20 months (range, 1 to 20 mo; mean, 9 mo), and 1 patient had no evidence of disease at 38 months. PESCC represents a unique, clinically aggressive subtype of endometrial cancer with TP53 and CDKN2A comutations. This characteristic profile, which is similar to HPV-independent squamous cell carcinoma of the vulva, is distinct from endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma and can be used to distinguish PESCC from those mimics even when cervical involvement is present. Diagnostic criteria for PESCC should be relaxed to allow for cervical involvement when other pathologic features are consistent with, and ancillary techniques are supportive of classification as such.

Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis.

Nature

2017 Aug 16

Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wiedenmann B, Nusse R, Amieva MR, Meyer TF.
PMID: 28813421 | DOI: 10.1038/nature23642

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

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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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