Probes for INS

ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

Probes for INS (4638)
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Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C

Immunity

2022 Apr 12

Zhao, M;Ren, K;Xiong, X;Xin, Y;Zou, Y;Maynard, JC;Kim, A;Battist, AP;Koneripalli, N;Wang, Y;Chen, Q;Xin, R;Yang, C;Huang, R;Yu, J;Huang, Z;Zhang, Z;Wang, H;Wang, D;Xiao, Y;Salgado, OC;Jarjour, NN;Hogquist, KA;Revelo, XS;Burlingame, AL;Gao, X;von Moltke, J;Lin, Z;Ruan, HB;
PMID: 35385697 | DOI: 10.1016/j.immuni.2022.03.009

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Single-neuron projectome of mouse prefrontal cortex

Nature neuroscience

2022 Apr 01

Gao, L;Liu, S;Gou, L;Hu, Y;Liu, Y;Deng, L;Ma, D;Wang, H;Yang, Q;Chen, Z;Liu, D;Qiu, S;Wang, X;Wang, D;Wang, X;Ren, B;Liu, Q;Chen, T;Shi, X;Yao, H;Xu, C;Li, CT;Sun, Y;Li, A;Luo, Q;Gong, H;Xu, N;Yan, J;
PMID: 35361973 | DOI: 10.1038/s41593-022-01041-5

Prefrontal cortex (PFC) is the cognitive center that integrates and regulates global brain activity. However, the whole-brain organization of PFC axon projections remains poorly understood. Using single-neuron reconstruction of 6,357 mouse PFC projection neurons, we identified 64 projectome-defined subtypes. Each of four previously known major cortico-cortical subnetworks was targeted by a distinct group of PFC subtypes defined by their first-order axon collaterals. Further analysis unraveled topographic rules of soma distribution within PFC, first-order collateral branch point-dependent target selection and terminal arbor distribution-dependent target subdivision. Furthermore, we obtained a high-precision hierarchical map within PFC and three distinct functionally related PFC modules, each enriched with internal recurrent connectivity. Finally, we showed that each transcriptome subtype corresponds to multiple projectome subtypes found in different PFC subregions. Thus, whole-brain single-neuron projectome analysis reveals organization principles of axon projections within and outside PFC and provides the essential basis for elucidating neuronal connectivity underlying diverse PFC functions.

Decomposing a deterministic path to mesenchymal niche formation by two intersecting morphogen gradients

Developmental cell

2022 Apr 25

Qu, R;Gupta, K;Dong, D;Jiang, Y;Landa, B;Saez, C;Strickland, G;Levinsohn, J;Weng, PL;Taketo, MM;Kluger, Y;Myung, P;
PMID: 35421372 | DOI: 10.1016/j.devcel.2022.03.011

Organ formation requires integrating signals to coordinate proliferation, specify cell fates, and shape tissue. Tracing these events and signals remains a challenge, as intermediate states across many critical transitions are unresolvable over real time and space. Here, we designed a unique computational approach to decompose a non-linear differentiation process into key components to resolve the signals and cell behaviors that drive a rapid transition, using the hair follicle dermal condensate as a model. Combining scRNA sequencing with genetic perturbation, we reveal that proliferative Dkk1+ progenitors transiently amplify to become quiescent dermal condensate cells by the mere spatiotemporal patterning of Wnt/β-catenin and SHH signaling gradients. Together, they deterministically coordinate a rapid transition from proliferation to quiescence, cell fate specification, and morphogenesis. Moreover, genetically repatterning these gradients reproduces these events autonomously in "slow motion" across more intermediates that resolve the process. This analysis unravels two morphogen gradients that intersect to coordinate events of organogenesis.

Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation

Nature communications

2022 Mar 25

Wang, LJ;Chen, CP;Lee, YS;Ng, PS;Chang, GD;Pao, YH;Lo, HF;Peng, CH;Cheong, ML;Chen, H;
PMID: 35338152 | DOI: 10.1038/s41467-022-29312-6

The combination of EGF, CHIR99021, A83-01, SB431542, VPA, and Y27632 (EGF/CASVY) facilitates the derivation of trophoblast stem (TS) cells from human blastocysts and first-trimester, but not term, cytotrophoblasts. The mechanism underlying this chemical induction of TS cells remains elusive. Here we demonstrate that the induction efficiency of cytotrophoblast is determined by functional antagonism of the placental transcription factor GCM1 and the stemness regulator ΔNp63α. ΔNp63α reduces GCM1 transcriptional activity, whereas GCM1 inhibits ΔNp63α oligomerization and autoregulation. EGF/CASVY cocktail activates ΔNp63α, thereby partially inhibiting GCM1 activity and reverting term cytotrophoblasts into stem cells. By applying hypoxia condition, we can further reduce GCM1 activity and successfully induce term cytotrophoblasts into TS cells. Consequently, we identify mitochondrial creatine kinase 1 (CKMT1) as a key GCM1 target crucial for syncytiotrophoblast differentiation and reveal decreased CKMT1 expression in preeclampsia. Our study delineates the molecular underpinnings of trophoblast stemness and differentiation and an efficient method to establish TS cells from term placentas.

Extracellular mechanical forces drive endocardial cell volume decrease during zebrafish cardiac valve morphogenesis

Developmental cell

2022 Mar 14

Vignes, H;Vagena-Pantoula, C;Prakash, M;Fukui, H;Norden, C;Mochizuki, N;Jug, F;Vermot, J;
PMID: 35245444 | DOI: 10.1016/j.devcel.2022.02.011

Organ morphogenesis involves dynamic changes of tissue properties while cells adapt to their mechanical environment through mechanosensitive pathways. How mechanical cues influence cell behaviors during morphogenesis remains unclear. Here, we studied the formation of the zebrafish atrioventricular canal (AVC) where cardiac valves develop. We show that the AVC forms within a zone of tissue convergence associated with the increased activation of the actomyosin meshwork and cell-orientation changes. We demonstrate that tissue convergence occurs with a reduction of cell volume triggered by mechanical forces and the mechanosensitive channel TRPP2/TRPV4. Finally, we show that the extracellular matrix component hyaluronic acid controls cell volume changes. Together, our data suggest that multiple force-sensitive signaling pathways converge to modulate cell volume. We conclude that cell volume reduction is a key cellular feature activated by mechanotransduction during cardiovascular morphogenesis. This work further identifies how mechanical forces and extracellular matrix influence tissue remodeling in developing organs.

SCAMPR: Single-Cell Automated Multiplex Pipeline for RNA Quantification and Spatial Mapping

SSRN Electronic Journal

2022 Mar 24

Ali Marandi Ghoddousi, R;Magalong, V;Kamitakahara, A;Levitt, P;
| DOI: 10.2139/ssrn.4064105

Spatial gene expression, achieved classically through _in situ_ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for the visualization of multiple mRNAs at single-cell resolution, greatly expanding the ability to link gene expression to tissue topography. Yet, methods for efficient and accurate quantification and analysis of high dimensional _in situ_- hybridization are limited. To this end, the Single-Cell Automated Multiplex Pipeline for RNA (SCAMPR) was developed, facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low and high abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on differential gene expression by vagal neuron subtypes.

The spatial transcriptomic landscape of the healing mouse intestine following damage

Nature communications

2022 Feb 11

Parigi, SM;Larsson, L;Das, S;Ramirez Flores, RO;Frede, A;Tripathi, KP;Diaz, OE;Selin, K;Morales, RA;Luo, X;Monasterio, G;Engblom, C;Gagliani, N;Saez-Rodriguez, J;Lundeberg, J;Villablanca, EJ;
PMID: 35149721 | DOI: 10.1038/s41467-022-28497-0

The intestinal barrier is composed of a complex cell network defining highly compartmentalized and specialized structures. Here, we use spatial transcriptomics to define how the transcriptomic landscape is spatially organized in the steady state and healing murine colon. At steady state conditions, we demonstrate a previously unappreciated molecular regionalization of the colon, which dramatically changes during mucosal healing. Here, we identified spatially-organized transcriptional programs defining compartmentalized mucosal healing, and regions with dominant wired pathways. Furthermore, we showed that decreased p53 activation defined areas with increased presence of proliferating epithelial stem cells. Finally, we mapped transcriptomics modules associated with human diseases demonstrating the translational potential of our dataset. Overall, we provide a publicly available resource defining principles of transcriptomic regionalization of the colon during mucosal healing and a framework to develop and progress further hypotheses.

SRSF1 governs progenitor-specific alternative splicing to maintain adult epithelial tissue homeostasis and renewal

Developmental cell

2022 Feb 22

Yu, T;Cazares, O;Tang, AD;Kim, HY;Wald, T;Verma, A;Liu, Q;Barcellos-Hoff, MH;Floor, SN;Jung, HS;Brooks, AN;Klein, OD;
PMID: 35202586 | DOI: 10.1016/j.devcel.2022.01.011

Alternative splicing generates distinct mRNA variants and is essential for development, homeostasis, and renewal. Proteins of the serine/arginine (SR)-rich splicing factor family are major splicing regulators that are broadly required for organ development as well as cell and organism viability. However, how these proteins support adult organ function remains largely unknown. Here, we used the continuously growing mouse incisor as a model to dissect the functions of the prototypical SR family protein SRSF1 during tissue homeostasis and renewal. We identified an SRSF1-governed alternative splicing network that is specifically required for dental proliferation and survival of progenitors but dispensable for the viability of differentiated cells. We also observed a similar progenitor-specific role of SRSF1 in the small intestinal epithelium, indicating a conserved function of SRSF1 across adult epithelial tissues. Thus, our findings define a regulatory mechanism by which SRSF1 specifically controls progenitor-specific alternative splicing events to support adult tissue homeostasis and renewal.

BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states

Cell reports

2022 Mar 01

Beumer, J;Puschhof, J;Yengej, FY;Zhao, L;Martinez-Silgado, A;Blotenburg, M;Begthel, H;Boot, C;van Oudenaarden, A;Chen, YG;Clevers, H;
PMID: 35235783 | DOI: 10.1016/j.celrep.2022.110438

Intestinal epithelial cells derive from stem cells at the crypt base and travel along the crypt-villus axis to die at the villus tip. The two dominant villus epithelial cell types, absorptive enterocytes and mucous-secreting goblet cells, are mature when they exit crypts. Murine enterocytes switch functional cell states during migration along the villus. Here, we ask whether this zonation is driven by the bone morphogenetic protein (BMP) gradient, which increases toward the villus. Using human intestinal organoids, we show that BMP signaling controls the expression of zonated genes in enterocytes. We find that goblet cells display similar zonation involving antimicrobial genes. Using an inducible Bmpr1a knockout mouse model, we confirm that BMP controls these zonated genes in vivo. Our findings imply that local manipulation of BMP signal strength may be used to reset the enterocyte "rheostat" of carbohydrate versus lipid uptake and to control the antimicrobial response through goblet cells.

Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing

PLoS biology

2022 Jan 01

Jasso, GJ;Jaiswal, A;Varma, M;Laszewski, T;Grauel, A;Omar, A;Silva, N;Dranoff, G;Porter, JA;Mansfield, K;Cremasco, V;Regev, A;Xavier, RJ;Graham, DB;
PMID: 35085231 | DOI: 10.1371/journal.pbio.3001532

Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.

Structure-Based Design of a Chemical Probe Set for the 5-HT5A Serotonin Receptor

Journal of medicinal chemistry

2022 Feb 23

Levit Kaplan, A;Strachan, RT;Braz, JM;Craik, V;Slocum, S;Mangano, T;Amabo, V;O'Donnell, H;Lak, P;Basbaum, AI;Roth, BL;Shoichet, BK;
PMID: 35195401 | DOI: 10.1021/acs.jmedchem.1c02031

The 5-HT5A receptor (5-HT5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT5AR homology model identified 5 mid-μM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT5AR activity revealed that 5-HT5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT5AR.

Towards Tabula Gallus

International journal of molecular sciences

2022 Jan 06

Yamagata, M;
PMID: 35054796 | DOI: 10.3390/ijms23020613

The Tabula Gallus is a proposed project that aims to create a map of every cell type in the chicken body and chick embryos. Chickens (Gallus gallus) are one of the most recognized model animals that recapitulate the development and physiology of mammals. The Tabula Gallus will generate a compendium of single-cell transcriptome data from Gallus gallus, characterize each cell type, and provide tools for the study of the biology of this species, similar to other ongoing cell atlas projects (Tabula Muris and Tabula Sapiens/Human Cell Atlas for mice and humans, respectively). The Tabula Gallus will potentially become an international collaboration between many researchers. This project will be useful for the basic scientific study of Gallus gallus and other birds (e.g., cell biology, molecular biology, developmental biology, neuroscience, physiology, oncology, virology, behavior, ecology, and evolution). It will eventually be beneficial for a better understanding of human health and diseases.

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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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