Single-cell RNA sequencing to study vascular diversity and function

Current opinion in hematology

2021 May 01

Ma, F;Hernandez, GE;Romay, M;Iruela-Arispe, ML;
PMID: 33714967 | DOI: 10.1097/MOH.0000000000000651

Single-cell RNA sequencing (scRNA-seq) can capture the transcriptional profile of thousands of individual cells concurrently from complex tissues and with remarkable resolution. Either with the goal of seeking information about distinct cell subtypes or responses to a stimulus, the approach has provided robust information and promoted impressive advances in cardiovascular research. The goal of this review is to highlight strategies and approaches to leverage this technology and bypass potential caveats related to evaluation of the vascular cells. As the most recent technological development, details associated with experimental strategies, analysis, and interpretation of scRNA-seq data are still being discussed and scrutinized by investigators across the vascular field. Compilation of this information is valuable for those using the technology but particularly important to those about to start utilizing scRNA-seq to seek transcriptome information of vascular cells. As our field progresses to catalog transcriptomes from distinct vascular beds, it is undeniable that scRNA-seq technology is here to stay. Sharing approaches to improve the quality of cell dissociation procedures, analysis, and a consensus of best practices is critical as information from this powerful experimental platform continues to emerge.

Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon

Neuron

2021 Apr 01

McElvain, LE;Chen, Y;Moore, JD;Brigidi, GS;Bloodgood, BL;Lim, BK;Costa, RM;Kleinfeld, D;
PMID: 33823137 | DOI: 10.1016/j.neuron.2021.03.017

Basal ganglia play a central role in regulating behavior, but the organization of their outputs to other brain areas is incompletely understood. We investigate the largest output nucleus, the substantia nigra pars reticulata (SNr), and delineate the organization and physiology of its projection populations in mice. Using genetically targeted viral tracing and whole-brain anatomical analysis, we identify over 40 SNr targets that encompass a roughly 50-fold range of axonal densities. Retrograde tracing from the volumetrically largest targets indicates that the SNr contains segregated subpopulations that differentially project to functionally distinct brain stem regions. These subpopulations are electrophysiologically specialized and topographically organized and collateralize to common diencephalon targets, including the motor and intralaminar thalamus as well as the pedunculopontine nucleus and the midbrain reticular formation. These findings establish that SNr signaling is organized as dense, parallel outputs to specific brain stem targets concurrent with extensive collateral branches that encompass the majority of SNr axonal boutons.

Identifying and characterizing virus-encoded circular RNAs

Methods (San Diego, Calif.)

2021 Mar 11

Tagawa, T;Kopardé, VN;Ziegelbauer, JM;
PMID: 33713796 | DOI: 10.1016/j.ymeth.2021.03.004

Circular forms of RNA were first discovered in plant viroids and later found in a variety of animal viruses. These circular RNAs lack free 5' and 3' ends, granting protection from exonucleases. This review is focused on the methods that are used to investigate virus-encoded circular RNAs. Using DNA viruses that are prevalent among human as examples, we begin with features of circular RNAs and the unique methods to enrich for circular RNAs. Next, we discuss the computational methods for RNA-sequencing analysis to discover new virus-encoded circular RNAs. Many strategies are similar to analyzing cellular RNAs, but some unique aspects of virus-encoded circular RNAs that are likely due to highly packed viral genomes and non-canonical use of splicing machinery, are described herein. We illustrate the various methods of validating expression of specific virus-encoded circular RNAs. Finally, we discuss novel methods to study functions of circular RNAs and the current technical challenges that remain for investigating virus-encoded circular RNAs.

Neuron-specific spinal cord translatomes reveal a neuropeptide code for mouse dorsal horn excitatory neurons

Scientific reports

2021 Mar 04

Das Gupta, RR;Scheurer, L;Pelczar, P;Wildner, H;Zeilhofer, HU;
PMID: 33664406 | DOI: 10.1038/s41598-021-84667-y

The spinal dorsal horn harbors a sophisticated and heterogeneous network of excitatory and inhibitory neurons that process peripheral signals encoding different sensory modalities. Although it has long been recognized that this network is crucial both for the separation and the integration of sensory signals of different modalities, a systematic unbiased approach to the use of specific neuromodulatory systems is still missing. Here, we have used the translating ribosome affinity purification (TRAP) technique to map the translatomes of excitatory glutamatergic (vGluT2+) and inhibitory GABA and/or glycinergic (vGAT+ or Gad67+) neurons of the mouse spinal cord. Our analyses demonstrate that inhibitory and excitatory neurons are not only set apart, as expected, by the expression of genes related to the production, release or re-uptake of their principal neurotransmitters and by genes encoding for transcription factors, but also by a differential engagement of neuromodulator, especially neuropeptide, signaling pathways. Subsequent multiplex in situ hybridization revealed eleven neuropeptide genes that are strongly enriched in excitatory dorsal horn neurons and display largely non-overlapping expression patterns closely adhering to the laminar and presumably also functional organization of the spinal cord grey matter.

Differential regulation of β-catenin-mediated transcription via N- and C-terminal co-factors governs identity of murine intestinal epithelial stem cells

Nature communications

2021 Mar 01

Borrelli, C;Valenta, T;Handler, K;Vélez, K;Gurtner, A;Moro, G;Lafzi, A;Roditi, LV;Hausmann, G;Arnold, IC;Moor, AE;Basler, K;
PMID: 33649334 | DOI: 10.1038/s41467-021-21591-9

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.

Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1

EMBO molecular medicine

2021 Feb 22

Sflomos, G;Battista, L;Aouad, P;De Martino, F;Scabia, V;Stravodimou, A;Ayyanan, A;Ifticene-Treboux, A;RLS, ;Bucher, P;Fiche, M;Ambrosini, G;Brisken, C;
PMID: 33616307 | DOI: 10.15252/emmm.202013180

Invasive lobular carcinoma (ILC) is the most frequent special histological subtype of breast cancer, typically characterized by loss of E-cadherin. It has clinical features distinct from other estrogen receptor-positive (ER+ ) breast cancers but the molecular mechanisms underlying its characteristic biology are poorly understood because we lack experimental models to study them. Here, we recapitulate the human disease, including its metastatic pattern, by grafting ILC-derived breast cancer cell lines, SUM-44 PE and MDA-MB-134-VI cells, into the mouse milk ducts. Using patient-derived intraductal xenografts from lobular and non-lobular ER+ HER2- tumors to compare global gene expression, we identify extracellular matrix modulation as a lobular carcinoma cell-intrinsic trait. Analysis of TCGA patient datasets shows matrisome signature is enriched in lobular carcinomas with overexpression of elastin, collagens, and the collagen modifying enzyme LOXL1. Treatment with the pan LOX inhibitor BAPN and silencing of LOXL1 expression decrease tumor growth, invasion, and metastasis by disrupting ECM structure resulting in decreased ER signaling. We conclude that LOXL1 inhibition is a promising therapeutic strategy for ILC.

COCO/DAND5 inhibits developmental and pathological ocular angiogenesis

EMBO molecular medicine

2021 Feb 15

Popovic, N;Hooker, E;Barabino, A;Flamier, A;Provost, F;Buscarlet, M;Bernier, G;Larrivée, B;
PMID: 33587337 | DOI: 10.15252/emmm.202012005

Neovascularization contributes to multiple visual disorders including age-related macular degeneration (AMD) and retinopathy of prematurity. Current therapies for treating ocular angiogenesis are centered on the inhibition of vascular endothelial growth factor (VEGF). While clinically effective, some AMD patients are refractory or develop resistance to anti-VEGF therapies and concerns of increased risks of developing geographic atrophy following long-term treatment have been raised. Identification of alternative pathways to inhibit pathological angiogenesis is thus important. We have identified a novel inhibitor of angiogenesis, COCO, a member of the Cerberus-related DAN protein family. We demonstrate that COCO inhibits sprouting, migration and cellular proliferation of cultured endothelial cells. Intravitreal injections of COCO inhibited retinal vascularization during development and in models of retinopathy of prematurity. COCO equally abrogated angiogenesis in models of choroidal neovascularization. Mechanistically, COCO inhibited TGFβ and BMP pathways and altered energy metabolism and redox balance of endothelial cells. Together, these data show that COCO is an inhibitor of retinal and choroidal angiogenesis, possibly representing a therapeutic option for the treatment of neovascular ocular diseases.

A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

Nature communications

2021 Feb 11

Zhu, Y;Feng, F;Hu, G;Wang, Y;Yu, Y;Zhu, Y;Xu, W;Cai, X;Sun, Z;Han, W;Ye, R;Qu, D;Ding, Q;Huang, X;Chen, H;Xu, W;Xie, Y;Cai, Q;Yuan, Z;Zhang, R;
PMID: 33574281 | DOI: 10.1038/s41467-021-21213-4

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei

Neuron

2021 Feb 06

Ma, T;Wong, SZH;Lee, B;Ming, GL;Song, H;
PMID: 33600763 | DOI: 10.1016/j.neuron.2021.01.026

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

Lung Mast Cells Have a High Constitutive Expression of Carboxypeptidase A3 mRNA That Is Independent from Granule-Stored CPA3

Cells

2021 Feb 03

Siddhuraj, P;Clausson, CM;Sanden, C;Alyamani, M;Kadivar, M;Marsal, J;Wallengren, J;Bjermer, L;Erjefält, JS;
PMID: 33546258 | DOI: 10.3390/cells10020309

The mast cell granule metalloprotease CPA3 is proposed to have important tissue homeostatic functions. However, the basal CPA3 mRNA and protein expression among mast cell populations has remained poorly investigated. Using a novel histology-based methodology that yields quantitative data on mRNA and protein expression at a single-cell level, the present study maps CPA3 mRNA and protein throughout the MCT and MCTC populations in healthy skin, gut and lung tissues. MCTC cells had both a higher frequency of CPA3 protein-containing cells and a higher protein-staining intensity than the MCT population. Among the tissues, skin MCs had highest CPA3 protein intensity. The expression pattern at the mRNA level was reversed. Lung mast cells had the highest mean CPA3 mRNA staining. Intriguingly, the large alveolar MCT population, that lack CPA3 protein, had uniquely high CPA3 mRNA intensity. A broader multi-tissue RNA analysis confirmed the uniquely high CPA3 mRNA quantities in the lung and corroborated the dissociation between chymase and CPA3 at the mRNA level. Taken together, our novel data suggest a hitherto underestimated contribution of mucosal-like MCT to baseline CPA3 mRNA production. The functional consequence of this high constitutive expression now reveals an important area for further research.

Single-cell atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to neuroblastoma phenotype

Proceedings of the National Academy of Sciences of the United States of America

2021 Feb 02

Hanemaaijer, ES;Margaritis, T;Sanders, K;Bos, FL;Candelli, T;Al-Saati, H;van Noesel, MM;Meyer-Wentrup, FAG;van de Wetering, M;Holstege, FCP;Clevers, H;
PMID: 33500353 | DOI: 10.1073/pnas.2022350118

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.

Noncanonical TGFβ Signaling Promotes Specialized Neuroretina Tip-Cell Sprouting and Blood-Retina Barrier Formation

SSRN Electronic Journal

2021 Jan 28

Zarkada, G;Howard, J;Xiao, X;Park, H;Bizou, M;Leclerc, S;Künzel, S;Cagnone, G;Joyal, J;Andelfinger, G;Eichmann, A;Dubrac, A;
| DOI: 10.2139/ssrn.3770937

Endothelial tip cells guiding tissue vascularization are primary targets for angiogenic therapies. Whether tip cells require differential signals to develop their complex branching patterns remained unknown. Here we show that tip cells invading the neuroretina (D-tip cells) are distinct from tip cells guiding the superficial plexus (S-tip cells). D-tip cells have a unique transcriptional signature, display blood-retina barrier properties and TGFβ signaling requirements. Endothelial deletion of TGFβ receptor I (ALK5) inhibits D-tip cell differentiation and diving vessels. ALK5 endothelial deficiency results in a dysfunctional S-like tip cell molecular signature, aberrant contractile pericytes, and hemorrhagic vascular malformations, while SMAD mutants do not develop this phenotype. Oxygen-induced retinopathy retinas exhibit S-like tip cells with increased Alk5 signaling, and _Alk5_ deletion impedes retina revascularization. Our data reveal stage-specific tip cell heterogeneity as a requirement for retinal vascular development and suggest that noncanonical-TGFβ signaling could improve retinal revascularization and neural function in ischemic retinopathy.

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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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