A subpopulation of embryonic microglia respond to maternal stress and influence nearby neural progenitors

Developmental cell

2021 Apr 19

Rosin, JM;Sinha, S;Biernaskie, J;Kurrasch, DM;
PMID: 33887203 | DOI: 10.1016/j.devcel.2021.03.018

The interplay between hypothalamic neurons and microglia as they integrate stressors to regulate homeostasis is of growing interest. We asked if microglia in the embryonic hypothalamus were likewise stress responsive and, if so, whether their precocious activation perturbs nearby neural stem cell (NSC) programs. We performed single-cell transcriptomics to define embryonic hypothalamic microglia heterogeneity and identified four microglial subsets, including a subpopulation adjacent to NSCs that was responsive to gestational cold stress. Stress exposure elevated CCL3 and CCL4 secretion, but only in male brains, and ex vivo CCL4 treatment of hypothalamic NSCs altered proliferation and differentiation. Concomitantly, gestational stress decreased PVN oxytocin neurons only in male embryos, which was reversed by microglia depletion. Adult offspring exposed to gestational stress displayed altered social behaviors, which was likewise microglia dependent, but only in males. Collectively, immature hypothalamic microglia play an unappreciated role in translating maternal stressors to sexually dimorphic perturbation of neurodevelopmental programs.

Promotion of cholangiocarcinoma growth by diverse cancer-associated fibroblast subpopulations

Cancer cell

2021 Apr 13

Affo, S;Nair, A;Brundu, F;Ravichandra, A;Bhattacharjee, S;Matsuda, M;Chin, L;Filliol, A;Wen, W;Song, X;Decker, A;Worley, J;Caviglia, JM;Yu, L;Yin, D;Saito, Y;Savage, T;Wells, RG;Mack, M;Zender, L;Arpaia, N;Remotti, HE;Rabadan, R;Sims, P;Leblond, AL;Weber, A;Riener, MO;Stockwell, BR;Gaublomme, J;Llovet, JM;Michalopoulos, GK;Seki, E;Sia, D;Chen, X;Califano, A;Schwabe, RF;
PMID: 33930309 | DOI: 10.1016/j.ccell.2021.03.012

Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.

Circular sisRNA identification and characterisation

Methods (San Diego, Calif.)

2021 Apr 08

Ng, AYE;Pek, JW;
PMID: 33838268 | DOI: 10.1016/j.ymeth.2021.04.002

Stable Intronic Sequence RNA (sisRNA) is a relatively new class of non-coding RNA. Found in many organisms, these sisRNA produced from their host genes are generally involved in regulatory roles, controlling gene expression at multiple levels through active involvement in regulatory feedback loops. Large scale identification of sisRNA via genome-wide RNA sequencing has been difficult, largely in part due to its low abundance. Done on its own, RNA sequencing often yields a large mass of information that is ironically uninformative; the potential sisRNA reads being masked by other highly abundant RNA species like ribosomal RNA and messenger RNA. In this review, we present a practical workflow for the enrichment of circular sisRNA through the use of transcriptionally quiescent systems, rRNA-depletion, and RNase R treatment prior to deep sequencing. This workflow allows circular sisRNA to be reliably detected. We also present various methods to experimentally validate the circularity and stability of the circular sisRNA identified, as well as a few methods for further functional characterisation.

Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Scientific reports

2021 Mar 24

Ma, J;Tran, G;Wan, AMD;Young, EWK;Kumacheva, E;Iscove, NN;Zandstra, PW;
PMID: 33762663 | DOI: 10.1038/s41598-021-86087-4

Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

Enhancer viruses for combinatorial cell-subclass-specific labeling

Neuron

2021 Mar 23

Graybuck, LT;Daigle, TL;Sedeño-Cortés, AE;Walker, M;Kalmbach, B;Lenz, GH;Morin, E;Nguyen, TN;Garren, E;Bendrick, JL;Kim, TK;Zhou, T;Mortrud, M;Yao, S;Siverts, LA;Larsen, R;Gore, BB;Szelenyi, ER;Trader, C;Balaram, P;van Velthoven, CTJ;Chiang, M;Mich, JK;Dee, N;Goldy, J;Cetin, AH;Smith, K;Way, SW;Esposito, L;Yao, Z;Gradinaru, V;Sunkin, SM;Lein, E;Levi, BP;Ting, JT;Zeng, H;Tasic, B;
PMID: 33789083 | DOI: 10.1016/j.neuron.2021.03.011

Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.

Hair loss caused by gain-of-function mutant TRPV3 is associated with premature differentiation of follicular keratinocytes

The Journal of investigative dermatology

2021 Mar 03

Song, Z;Chen, X;Zhao, Q;Stanic, V;Lin, Z;Yang, S;Chen, T;Chen, J;Yang, Y;
PMID: 33675791 | DOI: 10.1016/j.jid.2020.11.036

Gain-of-function mutations in the TRPV3 gene can cause Olmsted syndrome characterized by palmoplantar and periorificial keratoderma, itch, and hair loss. The mechanism underlying hair loss remains unclear. Here, we engineered an Olmsted syndrome mouse model by introducing the point mutation G568V to the corresponding Trpv3 locus in mice. These mice developed fully penetrant hair loss. The hair loss was associated with premature differentiation of follicular keratinocytes characterized by precocious degeneration of trichohyalin and keratins, increased production of deiminated proteins, elevated apoptosis, and attenuation of transcription regulators (Foxn1, Msx2, Dlx3, and Gata3) known to regulate hair follicle differentiation. These abnormalities occurred in the medial-proximal region of the inner root sheath and the hair shaft, where Trpv3 is highly expressed, and correlated with impaired formation of the hair canal and the hair shaft. The mutant Trpv3 mice also exhibited increased proliferation in the outer root sheath, accelerated hair cycle, reduction of hair follicle stem cells, and miniaturization of regenerated hair follicles. Findings from this study suggest that precocious maturation of postmitotic follicular keratinocytes drives hair loss in Olmsted syndrome patients.

CD34T+ Humanized Mouse Model to Study Mucosal HIV-1 Transmission and Prevention

Vaccines

2021 Feb 27

Vanshylla, K;Held, K;Eser, TM;Gruell, H;Kleipass, F;Stumpf, R;Jain, K;Weiland, D;Münch, J;Grüttner, B;Geldmacher, C;Klein, F;
PMID: 33673566 | DOI: 10.3390/vaccines9030198

Humanized mice are critical for HIV-1 research, but humanized mice generated from cord blood are inefficient at mucosal HIV-1 transmission. Most mucosal HIV-1 transmission studies in mice require fetal tissue-engraftment, the use of which is highly restricted or prohibited. We present a fetal tissue-independent model called CD34T+ with enhanced human leukocyte levels in the blood and improved T cell homing to the gut-associated lymphoid tissue. CD34T+ mice are highly permissive to intra-rectal HIV-1 infection and also show normal env diversification in vivo despite high viral replication. Moreover, mucosal infection in CD34T+ mice can be prevented by infusion of broadly neutralizing antibodies. CD34T+ mice can be rapidly and easily generated using only cord blood cells and do not require any complicated surgical procedures for the humanization process. Therefore, CD34T+ mice provide a novel platform for mucosal HIV-1 transmission studies as well as rapid in vivo testing of novel prevention molecules against HIV-1.

An organoid-based organ-repurposing approach to treat short bowel syndrome

Nature

2021 Feb 24

Sugimoto, S;Kobayashi, E;Fujii, M;Ohta, Y;Arai, K;Matano, M;Ishikawa, K;Miyamoto, K;Toshimitsu, K;Takahashi, S;Nanki, K;Hakamata, Y;Kanai, T;Sato, T;
PMID: 33627870 | DOI: 10.1038/s41586-021-03247-2

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.

SOX9-COL9A3-dependent regulation of choroid plexus epithelial polarity governs blood-cerebrospinal fluid barrier integrity

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

2021 Feb 09

Vong, KI;Ma, TC;Li, B;Leung, TCN;Nong, W;Ngai, SM;Hui, JHL;Jiang, L;Kwan, KM;
PMID: 33526661 | DOI: 10.1073/pnas.2009568118

The choroid plexus (CP) is an extensively vascularized neuroepithelial tissue that projects into the brain ventricles. The restriction of transepithelial transport across the CP establishes the blood-cerebrospinal fluid (CSF) barrier that is fundamental to the homeostatic regulation of the central nervous system microenvironment. However, the molecular mechanisms that control this process remain elusive. Here we show that the genetic ablation of Sox9 in the hindbrain CP results in a hyperpermeable blood-CSF barrier that ultimately upsets the CSF electrolyte balance and alters CSF protein composition. Mechanistically, SOX9 is required for the transcriptional up-regulation of Col9a3 in the CP epithelium. The reduction of Col9a3 expression dramatically recapitulates the blood-CSF barrier defects of Sox9 mutants. Loss of collagen IX severely disrupts the structural integrity of the epithelial basement membrane in the CP, leading to progressive loss of extracellular matrix components. Consequently, this perturbs the polarized microtubule dynamics required for correct orientation of apicobasal polarity and thereby impedes tight junction assembly in the CP epithelium. Our findings reveal a pivotal cascade of SOX9-dependent molecular events that is critical for construction of the blood-CSF barrier.

Systematic analysis of purified astrocytes after SCI unveils Zeb2os function during astrogliosis

Cell reports

2021 Feb 02

Wei, H;Wu, X;You, Y;Duran, RC;Zheng, Y;Narayanan, KL;Hai, B;Li, X;Tallapragada, N;Prajapati, TJ;Kim, DH;Deneen, B;Cao, QL;Wu, JQ;
PMID: 33535036 | DOI: 10.1016/j.celrep.2021.108721

Spinal cord injury (SCI) is one of the most devastating neural injuries without effective therapeutic solutions. Astrocytes are the predominant component of the scar. Understanding the complex contributions of reactive astrocytes to SCI pathophysiologies is fundamentally important for developing therapeutic strategies. We have studied the molecular changes in the injury environment and the astrocyte-specific responses by astrocyte purification from injured spinal cords from acute to chronic stages. In addition to protein-coding genes, we have systematically analyzed the expression profiles of long non-coding RNAs (lncRNAs) (>200 bp), which are regulatory RNAs that play important roles in the CNS. We have identified a highly conserved lncRNA, Zeb2os, and demonstrated using functional assays that it plays an important role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis. These studies provide valuable insights into the molecular basis of reactive astrogliosis and fill the knowledge gap regarding the function(s) of lncRNAs in astrogliosis and SCI.

A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells

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

2021 Jan 26

Murakami, K;Terakado, Y;Saito, K;Jomen, Y;Takeda, H;Oshima, M;Barker, N;
PMID: 33479180 | DOI: 10.1073/pnas.2016806118

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3β, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

Dermal adipose tissue secretes HGF to promote human hair growth and pigmentation

The Journal of investigative dermatology

2021 Jan 22

Nicu, C;O'Sullivan, JDB;Ramos, R;Timperi, L;Lai, T;Farjo, N;Farjo, B;Pople, J;Bhogal, R;Hardman, JA;Plikus, MV;Ansell, DM;Paus, R;
PMID: 33493531 | DOI: 10.1016/j.jid.2020.12.019

Hair follicles (HFs) are immersed within dermal white adipose tissue (dWAT), yet human adipocyte-HF communication remains unexplored. Therefore, we investigated how perifollicular adipocytes affect the physiology of organ-cultured human anagen scalp HFs. Quantitative (immuno-)histomorphometry, microCT and transmission electron microscopy showed that the number and size of perifollicular adipocytes declined during anagen-catagen transition, whilst fluorescence lifetime imaging revealed increased lipid oxidation in adipocytes surrounding the bulge/sub-bulge region. Ex vivo, dWAT significantly stimulated hair matrix keratinocyte proliferation and HF pigmentation. Both dWAT pericytes and PREF1/DLK1+ adipocyte progenitors secreted hepatocyte growth factor (HGF) during human HF-dWAT co-culture, for which the c-Met receptor is expressed in the hair matrix and dermal papilla. These effects were abrogated by an HGF-neutralising antibody, and reproduced using recombinant HGF. Laser capture microdissection-based microarray analysis of the hair matrix showed that dWAT-derived HGF up-regulated KRT27, KRT73, KRT75, KRT84, KRT86 and TCHH. Mechanistically, HGF stimulated Wnt/β-catenin activity in the HM by inhibiting SFRP1 in the dermal papilla, up-regulating matrix AXIN2, LEF1, WNT6 and WNT10B expression. Our study demonstrates that dWAT regulates human hair growth and pigmentation via HGF secretion, and thus identifies important, molecular and cellular targets for therapeutic intervention in disorders of human hair growth and pigmentation.

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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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