Cell-specific RNA purification to study translatomes of mouse central nervous system

STAR protocols

2022 Jun 17

Bravo-Ferrer, I;Khakh, BS;Díaz-Castro, B;
PMID: 35620074 | DOI: 10.1016/j.xpro.2022.101397

Cell-specific RNA sequencing has revolutionized the study of cell biology. Here, we present a protocol to assess cell-specific translatomes of genetically targeted cell types. We focus on astrocytes and describe RNA purification using RiboTag tools. Unlike single-cell RNA sequencing, this approach allows high sequencing depth to detect low expression genes, and the exploration of RNAs translated in subcellular compartments. Furthermore, it avoids underestimation of transcripts from cells susceptible to cell isolation procedures. The protocol can be applied to a variety of cell types. For complete details on the use and execution of this protocol, please refer to Chai et al. (2017), Díaz-Castro et al. (2021), Díaz-Castro et al. (2019), Srinivasan et al. (2016), and Yu et al. (2018).

Studying of Molecular Regulation of Developmental Processes of Lower Metazoans Exemplified by Cnidaria Using High-Throughput Sequencing

Biochemistry (Moscow)

2022 Mar 01

Erofeeva, T;Grigorenko, A;Gusev, F;Kosevich, I;Rogaev, E;
| DOI: 10.1134/S0006297922030075

A unique set of features and characteristics of species of the Cnidaria phylum is the one reason that makes them a model for a various studies. The plasticity of a life cycle and the processes of cell differentiation and development of an integral multicellular organism associated with it are of a specific scientific interest. A new stage of development of molecular genetic methods, including methods for high-throughput genome, transcriptome, and epigenome sequencing, both at the level of the whole organism and at the level of individual cells, makes it possible to obtain a detailed picture of the development of these animals. This review examines some modern approaches and advances in the reconstruction of the processes of ontogenesis of cnidarians by studying the regulatory signal transduction pathways and their interactions.

GABA-receptive microglia selectively sculpt developing inhibitory circuits

Cell

2021 Jul 22

Favuzzi, E;Huang, S;Saldi, GA;Binan, L;Ibrahim, LA;Fernández-Otero, M;Cao, Y;Zeine, A;Sefah, A;Zheng, K;Xu, Q;Khlestova, E;Farhi, SL;Bonneau, R;Datta, SR;Stevens, B;Fishell, G;
PMID: 34233165 | DOI: 10.1016/j.cell.2021.06.018

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.

Embryo-scale, single-cell spatial transcriptomics

Science (New York, N.Y.)

2021 Jul 02

Srivatsan, SR;Regier, MC;Barkan, E;Franks, JM;Packer, JS;Grosjean, P;Duran, M;Saxton, S;Ladd, JJ;Spielmann, M;Lois, C;Lampe, PD;Shendure, J;Stevens, KR;Trapnell, C;
PMID: 34210887 | DOI: 10.1126/science.abb9536

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.

A constant pool of Lgr5+ intestinal stem cells is required for intestinal homeostasis

Cell reports

2021 Jan 26

Tan, SH;Phuah, P;Tan, LT;Yada, S;Goh, J;Tomaz, LB;Chua, M;Wong, E;Lee, B;Barker, N;
PMID: 33503423 | DOI: 10.1016/j.celrep.2020.108633

Lgr5+ crypt base columnar cells, the operational intestinal stem cells (ISCs), are thought to be dispensable for small intestinal (SI) homeostasis. Using a Lgr5-2A-DTR (diphtheria toxin receptor) model, which ablates Lgr5+ cells with near-complete efficiency and retains endogenous levels of Lgr5 expression, we show that persistent depletion of Lgr5+ ISCs in fact compromises SI epithelial integrity and reduces epithelial turnover in vivo. In vitro, Lgr5-2A-DTR SI organoids are unable to establish or survive when Lgr5+ ISCs are continuously eliminated by adding DT to the media. However, transient exposure to DT at the start of culture allows organoids to form, and the rate of outgrowth reduces with the increasing length of DT presence. Our results indicate that intestinal homeostasis requires a constant pool of Lgr5+ ISCs, which is supplied by rapidly reprogrammed non-Lgr5+ crypt populations when preexisting Lgr5+ ISCs are ablated.

T-Cell Receptor Repertoire Sequencing in the Era of Cancer Immunotherapy

Clinical cancer research : an official journal of the American Association for Cancer Research

2023 Mar 14

Frank, ML;Lu, K;Erdogan, C;Han, Y;Hu, J;Wang, T;Heymach, JV;Zhang, J;Reuben, A;
PMID: 36413126 | DOI: 10.1158/1078-0432.CCR-22-2469

T cells are integral components of the adaptive immune system, and their responses are mediated by unique T-cell receptors (TCR) that recognize specific antigens from a variety of biological contexts. As a result, analyzing the T-cell repertoire offers a better understanding of immune responses and of diseases like cancer. Next-generation sequencing technologies have greatly enabled the high-throughput analysis of the TCR repertoire. On the basis of our extensive experience in the field from the past decade, we provide an overview of TCR sequencing, from the initial library preparation steps to sequencing and analysis methods and finally to functional validation techniques. With regards to data analysis, we detail important TCR repertoire metrics and present several computational tools for predicting antigen specificity. Finally, we highlight important applications of TCR sequencing and repertoire analysis to understanding tumor biology and developing cancer immunotherapies.

Parvalbumin Gates Chronic Pain Through the Modulation of Tonic Firing in Inhibitory Neurons

Available at SSRN

2023 Jan 01

Qiu, H;Miraucourt, LS;Petitjean, H;Theriault, C;

Spinal cord dorsal horn (DH) inhibition is critical to the processing of sensory inputs, and its impairment leads to mechanical allodynia. How this decreased inhibition occurs and whether its restoration alleviates allodynic pain is poorly understood. Here, we show that the calcium (Ca2+)-binding protein, parvalbumin (PV), controls the activity of inhibitory PV-expressing neurons (PVNs) by enabling them to sustain high-frequency tonic firing patterns. Upon nerve injury, PVNs transition to adaptive firing and decrease their PV expression. Interestingly, decreased PV is necessary and sufficient to the development of mechanical allodynia and the transition of PVNs to adaptive firing. This transition of firing pattern is due to the recruitment of calcium-activated potassium (SK) channels and blocking them during chronic pain restores normal tonic firing. Our findings indicate that PV is essential to the firing activity of PVNs and in preventing allodynia, these observations may lead to novel strategies for chronic pain relief.

A single-cell transcriptional atlas reveals resident progenitor cell niche functions in TMJ disc development and injury

Nature communications

2023 Feb 14

Bi, R;Yin, Q;Li, H;Yang, X;Wang, Y;Li, Q;Fang, H;Li, P;Lyu, P;Fan, Y;Ying, B;Zhu, S;
PMID: 36788226 | DOI: 10.1038/s41467-023-36406-2

The biological characteristics of the temporomandibular joint disc involve complex cellular network in cell identity and extracellular matrix composition to modulate jaw function. The lack of a detailed characterization of the network severely limits the development of targeted therapies for temporomandibular joint-related diseases. Here we profiled single-cell transcriptomes of disc cells from mice at different postnatal stages, finding that the fibroblast population could be divided into chondrogenic and non-chondrogenic clusters. We also find that the resident mural cell population is the source of disc progenitors, characterized by ubiquitously active expression of the NOTCH3 and THY1 pathways. Lineage tracing reveals that Myh11+ mural cells coordinate angiogenesis during disc injury but lost their progenitor characteristics and ultimately become Sfrp2+ non-chondrogenic fibroblasts instead of Chad+ chondrogenic fibroblasts. Overall, we reveal multiple insights into the coordinated development of disc cells and are the first to describe the resident mural cell progenitor during disc injury.

Gut microbiota promotes stem cell differentiation through macrophage and mesenchymal niches in early postnatal development

Immunity

2022 Dec 13

Kim, JE;Li, B;Fei, L;Horne, R;Lee, D;Loe, AK;Miyake, H;Ayar, E;Kim, DK;Surette, MG;Philpott, DJ;Sherman, P;Guo, G;Pierro, A;Kim, TH;
PMID: 36473468 | DOI: 10.1016/j.immuni.2022.11.003

Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206+ subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206+ macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.

In mice and humans, brain microvascular contractility matures postnatally

Brain structure & function

2022 Nov 16

Slaoui, L;Gilbert, A;Rancillac, A;Delaunay-Piednoir, B;Chagnot, A;Gerard, Q;Letort, G;Mailly, P;Robil, N;Gelot, A;Lefebvre, M;Favier, M;Dias, K;Jourdren, L;Federici, L;Auvity, S;Cisternino, S;Vivien, D;Cohen-Salmon, M;Boulay, AC;
PMID: 36380034 | DOI: 10.1007/s00429-022-02592-w

Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.

Modulation of tissue resident memory T cells by glucocorticoids after acute cellular rejection in lung transplantation

The Journal of experimental medicine

2022 Apr 04

Snyder, ME;Moghbeli, K;Bondonese, A;Craig, A;Popescu, I;Fan, L;Tabib, T;Lafyatis, R;Chen, K;Trejo Bittar, HE;Lendermon, E;Pilewski, J;Johnson, B;Kilaru, S;Zhang, Y;Sanchez, PG;Alder, JK;Sims, PA;McDyer, JF;
PMID: 35285873 | DOI: 10.1084/jem.20212059

Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single-cell RNA and TCR sequencing on recipient-derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with rejection and compare them with T cells obtained from the same patients after treatment of rejection with high-dose systemic glucocorticoids. At the time of rejection, we found an oligoclonal expansion of cytotoxic CD8+ T cells that all persisted as tissue resident memory T cells after successful treatment. Persisting CD8+ allograft-resident T cells have reduced gene expression for cytotoxic mediators after therapy with glucocorticoids but accumulate around airways. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in situ expansion. We thus highlight the accumulation of cytotoxic, recipient-derived tissue resident memory T cells within the lung allograft that persist despite the administration of high-dose systemic glucocorticoids. The long-term clinical consequences of this persistence have yet to be characterized.

Epidermal growth factor-like domain protein 6 recombinant protein facilitates osteogenic differentiation in adipose stem cells via bone morphogenetic protein 2/recombinant mothers against decapentaplegic homolog 4 signaling pathway

Bioengineered

2022 Mar 01

Liu, H;Wang, X;
PMID: 35220882 | DOI: 10.1080/21655979.2022.2037380

Adipose-derived mesenchymal stem cells (ADSCs) are a class of pluripotent stem cells isolated from the adipose tissue; they can differentiate into osteoblasts after induction and play an important role in bone repair. EGFL6 protein is secreted by adipocytes and osteoblasts and can promote endothelial cell migration and angiogenesis. This study aimed to explore the effect of recombinant EGFL6 protein on the osteogenic differentiation of ADSCs. The cells were incubated with fluorescein isothiocyanate-conjugated antibodies and analyzed by flow cytometry. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic differentiation ability. mRNA expression was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Protein expression was determined using Western blotting. The osteogenic differentiation ability of ADSCs isolated from the adipose tissue was significantly weakened after EGFL6 knockdown; this ability was restored upon the addition of EGFL6 recombinant protein. BMP2 knockdown inhibited the effect of EGFL6 recombinant protein on osteogenic differentiation. EGFL6 recombinant protein promoted osteogenic differentiation of ADSCs through the BMP2/SMAD4 signaling pathway. This may provide a potential target for the osteogenic differentiation of ADSCs.

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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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