X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

Donkey Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor ?? 647 Conjugated

RRID:AB_2536183

Antibody ID

AB_2536183

Target Antigen

Rabbit IgG (H+L) rabbit

Vendor

Molecular Probes

Cat Num

A-31573 also A31573

Proper Citation

(Molecular Probes Cat# A-31573, RRID:AB_2536183)

Clonality

polyclonal antibody

Host Organism

donkey

Dual leucine zipper kinase is required for mechanical allodynia and microgliosis after nerve injury.

  • Wlaschin JJ
  • Elife
  • 2018 Jul 3

Literature context: RRID:AB_2536183 1:500 for immunostaining;


Abstract:

Neuropathic pain resulting from nerve injury can become persistent and difficult to treat but the molecular signaling responsible for its development remains poorly described. Here, we identify the neuronal stress sensor dual leucine zipper kinase (DLK; Map3k12) as a key molecule controlling the maladaptive pathways that lead to pain following injury. Genetic or pharmacological inhibition of DLK reduces mechanical hypersensitivity in a mouse model of neuropathic pain. Furthermore, DLK inhibition also prevents the spinal cord microgliosis that results from nerve injury and arises distant from the injury site. These striking phenotypes result from the control by DLK of a transcriptional program in somatosensory neurons regulating the expression of numerous genes implicated in pain pathogenesis, including the immune gene Csf1. Thus, activation of DLK is an early event, or even the master regulator, controlling a wide variety of pathways downstream of nerve injury that ultimately lead to chronic pain.

Funding information:
  • National Center for Complementary and Integrative Health - Intramural Research Program()
  • National Institute of Child Heath and Human Development - Intramural Research Program()
  • National Institutes of Health - Intramural Research Program - DDIR Innovation Award()
  • NCI NIH HHS - P30 CA33572(United States)

Restricted Presence of POU6F2 in Human Corneal Endothelial Cells Uncovered by Extension of the Promoter-level Expression Atlas.

  • Yoshihara M
  • EBioMedicine
  • 2018 Jul 9

Literature context: abbit IgG (RRID: AB_2534013 and RRID: AB_2536183; Life Technologies) at room tem


Abstract:

Corneal endothelial cells (CECs) are essential for maintaining the clarity of the cornea. Because CECs have limited proliferative ability, interest is growing in their potentially therapeutic regeneration from pluripotent stem cells. However, the molecular mechanisms of human CEC differentiation remain largely unknown. To determine the key regulators of CEC characteristics, here we generated a comprehensive promoter-level expression profile of human CECs, using cap analysis of gene expression (CAGE) with a single molecule sequencer. Integration with the FANTOM5 promoter-level expression atlas, which includes transcriptome profiles of various human tissues and cells, enabled us to identify 45 promoters at 28 gene loci that are specifically expressed in CECs. We further discovered that the expression of transcription factor POU class 6 homeobox 2 (POU6F2) is restricted to CECs, and upregulated during human CEC differentiation, suggesting that POU6F2 is pivotal to terminal differentiation of CECs. These CEC-specific promoters would be useful for the assessment of fully differentiated CECs derived from pluripotent stem cells. These findings promote the development of corneal regenerative medicine.

Funding information:
  • NIMH NIH HHS - R01 MH094714(United States)

Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males.

  • Sousa EJ
  • Cell Stem Cell
  • 2018 Jun 1

Literature context: Fisher Scientific Cat# A-31573, RRID:AB_2536183 Donkey Anti-Rat IgG (H+L) Highl


Abstract:

A hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.

Funding information:
  • NHLBI NIH HHS - HL085036(United States)

Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb.

  • Oboti L
  • eNeuro
  • 2018 Jun 19

Literature context: ti-rabbit (RRID:AB_2536183; all diluted 1:1000).


Abstract:

Interconnections between the olfactory bulb and the amygdala are a major pathway for triggering strong behavioral responses to a variety of odorants. However, while this broad mapping has been established, the patterns of amygdala feedback connectivity and the influence on olfactory circuitry remain unknown. Here, using a combination of neuronal tracing approaches, we dissect the connectivity of a cortical amygdala [posteromedial cortical nucleus (PmCo)] feedback circuit innervating the mouse accessory olfactory bulb. Optogenetic activation of PmCo feedback mainly results in feedforward mitral cell (MC) inhibition through direct excitation of GABAergic granule cells. In addition, LED-driven activity of corticofugal afferents increases the gain of MC responses to olfactory nerve stimulation. Thus, through corticofugal pathways, the PmCo likely regulates primary olfactory and social odor processing.

Funding information:
  • NIDA NIH HHS - R01 DA020140()
  • NIDCD NIH HHS - R01 DC012050()
  • PHS HHS - R01 A1030048(United States)

FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex.

  • Künzel U
  • Elife
  • 2018 Jun 13

Literature context: RRID:AB_2536183 dilution is described in


Abstract:

Many intercellular signals are synthesised as transmembrane precursors that are released by proteolytic cleavage ('shedding') from the cell surface. ADAM17, a membrane-tethered metalloprotease, is the primary shedding enzyme responsible for the release of the inflammatory cytokine TNFα and several EGF receptor ligands. ADAM17 exists in complex with the rhomboid-like iRhom proteins, which act as cofactors that regulate ADAM17 substrate shedding. Here we report that the poorly characterised FERM domain-containing protein FRMD8 is a new component of the iRhom2/ADAM17 sheddase complex. FRMD8 binds to the cytoplasmic N-terminus of iRhoms and is necessary to stabilise iRhoms and ADAM17 at the cell surface. In the absence of FRMD8, iRhom2 and ADAM17 are degraded via the endolysosomal pathway, resulting in the reduction of ADAM17-mediated shedding. We have confirmed the pathophysiological significance of FRMD8 in iPSC-derived human macrophages and mouse tissues, thus demonstrating its role in the regulated release of multiple cytokine and growth factor signals.

Funding information:
  • Boehringer Ingelheim Fonds - PhD Fellowship()
  • Horizon 2020 Framework Programme - 659166()
  • Medical Research Council - 1374214()
  • Medical Research Council - MC_EX_MR/N50192X/1()
  • NINDS NIH HHS - NS054042(United States)
  • Wellcome - 101035/Z/13/Z()
  • Wellcome - Oxford Wellcome Institutional Strategic Support Fund 121302()

How Diverse Retinal Functions Arise from Feedback at the First Visual Synapse.

  • Drinnenberg A
  • Neuron
  • 2018 Jun 15

Literature context: tibody Invitrogen Cat# A-31573; RRID:AB_2536183 Alexa fluor 488 donkey anti-rab


Abstract:

Many brain regions contain local interneurons of distinct types. How does an interneuron type contribute to the input-output transformations of a given brain region? We addressed this question in the mouse retina by chemogenetically perturbing horizontal cells, an interneuron type providing feedback at the first visual synapse, while monitoring the light-driven spiking activity in thousands of ganglion cells, the retinal output neurons. We uncovered six reversible perturbation-induced effects in the response dynamics and response range of ganglion cells. The effects were enhancing or suppressive, occurred in different response epochs, and depended on the ganglion cell type. A computational model of the retinal circuitry reproduced all perturbation-induced effects and led us to assign specific functions to horizontal cells with respect to different ganglion cell types. Our combined experimental and theoretical work reveals how a single interneuron type can differentially shape the dynamical properties of distinct output channels of a brain region.

Funding information:
  • European Research Council - 233083(International)

Iron Drives T Helper Cell Pathogenicity by Promoting RNA-Binding Protein PCBP1-Mediated Proinflammatory Cytokine Production.

  • Wang Z
  • Immunity
  • 2018 Jun 21

Literature context: Life Tech Cat# A31573; RRID:AB_2536183 Chemicals, Peptides, and Recomb


Abstract:

Iron deposition is frequently observed in human autoinflammatory diseases, but its functional significance is largely unknown. Here we showed that iron promoted proinflammatory cytokine expression in T cells, including GM-CSF and IL-2, via regulating the stability of an RNA-binding protein PCBP1. Iron depletion or Pcbp1 deficiency in T cells inhibited GM-CSF production by attenuating Csf2 3' untranslated region (UTR) activity and messenger RNA stability. Pcbp1 deficiency or iron uptake blockade in autoreactive T cells abolished their capacity to induce experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Mechanistically, intracellular iron protected PCBP1 protein from caspase-mediated proteolysis, and PCBP1 promoted messenger RNA stability of Csf2 and Il2 by recognizing UC-rich elements in the 3' UTRs. Our study suggests that iron accumulation can precipitate autoimmune diseases by promoting proinflammatory cytokine production. RNA-binding protein-mediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/G007802/1(United Kingdom)

Myoepithelial Cells of Submucosal Glands Can Function as Reserve Stem Cells to Regenerate Airways after Injury.

  • Tata A
  • Cell Stem Cell
  • 2018 May 3

Literature context: bbit IgG Invitrogen A31573 RRID:AB_2536183 Alexa Fluoro 488 Donkey anti-go


Abstract:

Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.

Funding information:
  • NHLBI NIH HHS - R00 HL127181()
  • NIDDK NIH HHS - DK59630(United States)
  • NIEHS NIH HHS - U01 ES017219()

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens.

  • Pan J
  • Cell Syst
  • 2018 May 23

Literature context: or 647 Conjugated Thermo Fisher RRID:AB_2536183 Goat anti-Mouse IgG (H+L) Cross


Abstract:

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.

Funding information:
  • NCI NIH HHS - U01 CA176058()
  • NIDDK NIH HHS - U24 DK 58768-01A1(United States)

Phosphatidylinositol-5-Phosphate 4-Kinases Regulate Cellular Lipid Metabolism By Facilitating Autophagy.

  • Lundquist MR
  • Mol. Cell
  • 2018 May 3

Literature context: uor 647 Thermo Cat# A-31573, RRID:AB_2536183 Donkey anti-Mouse IgG Alexa Flu


Abstract:

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a-/-Pip4k2b-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.

Funding information:
  • NCI NIH HHS - R35 CA197588()
  • NCI NIH HHS - U54 CA210184()
  • NCRR NIH HHS - UL1RR024128(United States)
  • NIGMS NIH HHS - R01 GM041890()

Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength.

  • Kelley KW
  • Neuron
  • 2018 Apr 18

Literature context: vitrogen Cat# A-31573, RRID:AB_2536183 Alexa donkey anti-rabbit 488 In


Abstract:

Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) around MNs in a VGLUT1-dependent manner. Loss of astrocyte-encoded Kir4.1 selectively altered FαMN size and function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation might uncouple symptoms of muscle weakness from MN cell death in diseases like ALS.

Funding information:
  • FIC NIH HHS - K01 TW000001(United States)

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

  • Guo L
  • Cell
  • 2018 Apr 19

Literature context: 47 Molecular Probes Cat# A31573 RRID:AB_2536183 Donkey anti-Mouse IgG Secondary


Abstract:

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-β2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-β1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-β2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-β2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

Funding information:
  • NCI NIH HHS - P01CA684841(United States)
  • NIGMS NIH HHS - R01 GM069909()
  • NIGMS NIH HHS - R01 GM099836()
  • NIGMS NIH HHS - T32 GM008275()
  • NIGMS NIH HHS - T32 GM071339()
  • NINDS NIH HHS - F31 NS079009()
  • NINDS NIH HHS - R01 NS081303()
  • NINDS NIH HHS - R01 NS087227()
  • NINDS NIH HHS - R21 NS090205()
  • NINDS NIH HHS - R21 NS094921()
  • NINDS NIH HHS - R21 NS100055()
  • NINDS NIH HHS - R35 NS097263()
  • NINDS NIH HHS - R35 NS097974()

Hippo Signaling Plays an Essential Role in Cell State Transitions during Cardiac Fibroblast Development.

  • Xiao Y
  • Dev. Cell
  • 2018 Apr 23

Literature context: RRID:AB_2536183 Donkey anti-Goat, Alexa Fluro 4


Abstract:

During development, progenitors progress through transition states. The cardiac epicardium contains progenitors of essential non-cardiomyocytes. The Hippo pathway, a kinase cascade that inhibits the Yap transcriptional co-factor, controls organ size in developing hearts. Here, we investigated Hippo kinases Lats1 and Lats2 in epicardial diversification. Epicardial-specific deletion of Lats1/2 was embryonic lethal, and mutant embryos had defective coronary vasculature remodeling. Single-cell RNA sequencing revealed that Lats1/2 mutant cells failed to activate fibroblast differentiation but remained in an intermediate cell state with both epicardial and fibroblast characteristics. Lats1/2 mutant cells displayed an arrested developmental trajectory with persistence of epicardial markers and expanded expression of Yap targets Dhrs3, an inhibitor of retinoic acid synthesis, and Dpp4, a protease that modulates extracellular matrix (ECM) composition. Genetic and pharmacologic manipulation revealed that Yap inhibits fibroblast differentiation, prolonging a subepicardial-like cell state, and promotes expression of matricellular factors, such as Dpp4, that define ECM characteristics.

Funding information:
  • NIAAA NIH HHS - R01 AA020401(United States)

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation.

  • Hofweber M
  • Cell
  • 2018 Apr 19

Literature context: ermo RRID:AB_2536183 Alexa 647 Donkey anti-goat Ther


Abstract:

Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-β2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration.

Funding information:
  • NIGMS NIH HHS - R01 GM48435(United States)

Spatial-Temporal Lineage Restrictions of Embryonic p63+ Progenitors Establish Distinct Stem Cell Pools in Adult Airways.

  • Yang Y
  • Dev. Cell
  • 2018 Mar 26

Literature context: 647 Thermo Fisher Cat# A-31573; RRID:AB_2536183 donkey anti-chicken; Alexa Fluo


Abstract:

Basal cells (BCs) are p63-expressing multipotent progenitors of skin, tracheoesophageal and urinary tracts. p63 is abundant in developing airways; however, it remains largely unclear how embryonic p63+ cells contribute to the developing and postnatal respiratory tract epithelium, and ultimately how they relate to adult BCs. Using lineage-tracing and functional approaches in vivo, we show that p63+ cells arising from the lung primordium are initially multipotent progenitors of airway and alveolar lineages but later become restricted proximally to generate the tracheal adult stem cell pool. In intrapulmonary airways, these cells are maintained immature to adulthood in bronchi, establishing a rare p63+Krt5- progenitor cell population that responds to H1N1 virus-induced severe injury. Intriguingly, this pool includes a CC10 lineage-labeled p63+Krt5- cell subpopulation required for a full H1N1-response. These data elucidate key aspects in the establishment of regionally distinct adult stem cell pools in the respiratory system, potentially with relevance to other organs.

Funding information:
  • Intramural NIH HHS - ZIA HL006151-02(United States)
  • NCI NIH HHS - R01 CA112403()
  • NCI NIH HHS - R01 CA193455()
  • NHLBI NIH HHS - R35 HL135834()
  • NIAID NIH HHS - HHSN272201400008C()

Developmental History Provides a Roadmap for the Emergence of Tumor Plasticity.

  • Tata PR
  • Dev. Cell
  • 2018 Mar 26

Literature context: it IgG Invitrogen Cat# A-31573; RRID:AB_2536183 Alexa Fluor 488 Donkey anti-goa


Abstract:

We show that the loss or gain of transcription factor programs that govern embryonic cell-fate specification is associated with a form of tumor plasticity characterized by the acquisition of alternative cell fates normally characteristic of adjacent organs. In human non-small cell lung cancers, downregulation of the lung lineage-specifying TF NKX2-1 is associated with tumors bearing features of various gut tissues. Loss of Nkx2-1 from murine alveolar, but not airway, epithelium results in conversion of lung cells to gastric-like cells. Superimposing oncogenic Kras activation enables further plasticity in both alveolar and airway epithelium, producing tumors that adopt midgut and hindgut fates. Conversely, coupling Nkx2-1 loss with foregut lineage-specifying SOX2 overexpression drives the formation of squamous cancers with features of esophageal differentiation. These findings demonstrate that elements of pathologic tumor plasticity mirror the normal developmental history of organs in that cancer cells acquire cell fates associated with developmentally related neighboring organs.

Funding information:
  • NCI NIH HHS - R01CA172025(United States)
  • NHLBI NIH HHS - K99 HL127181()
  • NHLBI NIH HHS - P30 HL101287()
  • NHLBI NIH HHS - R00 HL127181()
  • NHLBI NIH HHS - R01 HL118185()
  • NIGMS NIH HHS - T32 GM007205()

Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.

  • Tavano S
  • Neuron
  • 2018 Mar 21

Literature context: Fisher Scientific Cat# A-31573, RRID:AB_2536183 Donkey anti-Mouse IgG (H+L) Hig


Abstract:

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Funding information:
  • Intramural NIH HHS - ZIA BC010763-05(United States)

Graded Arrays of Spinal and Supraspinal V2a Interneuron Subtypes Underlie Forelimb and Hindlimb Motor Control.

  • Hayashi M
  • Neuron
  • 2018 Feb 21

Literature context: Life technologies Cat#A-31573; RRID:AB_2536183 Goat anti-guinea pig secondary


Abstract:

The spinal cord contains neural networks that enable regionally distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both forelimb and hindlimb movements, and here we identify two major types that emerge during development: type I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons and type II neurons that downregulate Chx10 and project to supraspinal structures. Types I and II V2a interneurons are arrayed in counter-gradients, and this network activates different patterns of motor output at cervical and lumbar levels. Single-cell RNA sequencing (RNA-seq) revealed type I and II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.

Funding information:
  • European Commission - Advanced Grant 294354(United States)
  • NIA NIH HHS - R01 AG036040(United States)

Reconstruction of the Human Colon Epithelium In Vivo.

  • Sugimoto S
  • Cell Stem Cell
  • 2018 Feb 1

Literature context: ermo Fisher Scientific A-31573, RRID:AB_2536183 Donkey polyclonal anti-goat IgG


Abstract:

Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.

Funding information:
  • NIGMS NIH HHS - 5T32 GM 7288-37(United States)

Radial Glial Fibers Promote Neuronal Migration and Functional Recovery after Neonatal Brain Injury.

  • Jinnou H
  • Cell Stem Cell
  • 2018 Jan 4

Literature context: gG (H+L) Invitrogen Cat#A31573; RRID:AB_2536183 Alexa Fluor 647 donkey anti-mou


Abstract:

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

Funding information:
  • NIDDK NIH HHS - R01 DK082659(United States)

Establishment of TUSMi003-A, an induced pluripotent stem cell (iPSC) line from a 62-year old Chinese Han patient with Alzheimer's disease with ApoE3/4 genetic background.

  • Wang Y
  • Stem Cell Res
  • 2018 Jan 13

Literature context: Fisher Scientific Cat# A-31573, RRID:AB_2536183


Abstract:

A 62-year old Chinese Han Alzheimer's disease (AD) female patient with ApoE3/4 genetic background donated her Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system was used to reprogrammed PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in AD.

Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes.

  • Kinboshi M
  • Front Mol Neurosci
  • 2018 Jan 24

Literature context: :200; Thermo Fisher scientific, RRID:AB_2536183) were, respectively, used for v


Abstract:

Inwardly rectifying potassium (Kir) 4.1 channels in astrocytes regulate neuronal excitability by mediating spatial potassium buffering. Although dysfunction of astrocytic Kir4.1 channels is implicated in the development of epileptic seizures, the functional mechanisms of Kir4.1 channels in modulating epileptogenesis remain unknown. We herein evaluated the effects of Kir4.1 inhibition (blockade and knockdown) on expression of brain-derived neurotrophic factor (BDNF), a key modulator of epileptogenesis, in the primary cultures of mouse astrocytes. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner. Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of small interfering RNA (siRNA) targeting Kir4.1 significantly increased the mRNA and protein levels of BDNF. The BDNF induction by Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but not by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125. The present results demonstrated that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may be linked to the development of epilepsy and other neuropsychiatric disorders.

Funding information:
  • NINDS NIH HHS - R56 NS021072(United States)

Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of β-amyloidosis.

  • Huynh TV
  • Neuron
  • 2017 Dec 6

Literature context: robes Cat# A-31573 also A31573; RRID:AB_2536183 Chemicals, Peptides, and Recomb


Abstract:

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer disease. Previous studies suggest that reduction of apoE levels through genetic manipulation can reduce Aβ pathology. However, it is not clear how reduction of apoE levels after birth would affect amyloid deposition. We utilize an antisense oligonucleotide (ASO) to reduce apoE expression in the brains of APP/PS1-21 mice homozygous for the APOE-ε4 or APOE-ε3 allele. ASO treatment starting after birth led to a significant decrease in Aβ pathology when assessed at 4 months. Interestingly, ASO treatment starting at the onset of amyloid deposition led to an increase in Aβ plaque size and a reduction in plaque-associated neuritic dystrophy with no change in overall plaque load. These results suggest that lowering apoE levels prior to plaque deposition can strongly affect the initiation of Aβ pathology while lowering apoE after Aβ seeding modulates plaque size and toxicity.

Funding information:
  • NIA NIH HHS - R01 AG047644()
  • NIGMS NIH HHS - R01GM105431(United States)
  • NIGMS NIH HHS - T32 GM007200()
  • NINDS NIH HHS - R01 NS034467()
  • NINDS NIH HHS - R01 NS090934()
  • NINDS NIH HHS - R37 NS034467()

Distinct projection targets define subpopulations of mouse brainstem vagal neurons that express the autism-associated MET receptor tyrosine kinase.

  • Kamitakahara A
  • J. Comp. Neurol.
  • 2017 Dec 15

Literature context: Fisher Scientific Cat# A-31573, RRID:AB_2536183), and/or Alexa Fluor® 647 Donke


Abstract:

Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: (a) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, (b) EGFP+ neurons in the medial DMV projecting to the stomach, and (b) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggests that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.

The Primate-Specific Gene TMEM14B Marks Outer Radial Glia Cells and Promotes Cortical Expansion and Folding.

  • Liu J
  • Cell Stem Cell
  • 2017 Nov 2

Literature context: G(H+L) Invitrogen Cat# A-31573; RRID:AB_2536183 Alexa Fluor 647Conjuated Affini


Abstract:

Human brain evolution is associated with expansion and folding of the neocortex. Increased diversity in neural progenitor (NP) populations (such as basally located radial glia [RG], which reside in an enlarged outer subventricular zone [OSVZ]) likely contributes to this evolutionary expansion, although their characteristics and relative contributions are only partially understood. Through single-cell transcriptional profiling of sorted human NP subpopulations, we identified the primate-specific TMEM14B gene as a marker of basal RG. Expression of TMEM14B in embryonic NPs induces cortical thickening and gyrification in postnatal mice. This is accompanied by SVZ expansion, the appearance of outer RG-like cells, and the proliferation of multiple NP subsets, with proportional increases in all cortical layers and normal lamination. TMEM14B drives NP proliferation by increasing the phosphorylation and nuclear translocation of IQGAP1, which in turn promotes G1/S cell cycle transitions. These data show that a single primate-specific gene can drive neurodevelopmental changes that contribute to brain evolution.

Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway.

  • Le Guerroué F
  • Mol. Cell
  • 2017 Nov 16

Literature context: ogies Cat# A-31573 also A31573; RRID:AB_2536183 Chemicals, Peptides, and Recomb


Abstract:

Autophagy allows the degradation of cytosolic endogenous and exogenous material in the lysosome. Substrates are engulfed by double-membrane vesicles, coined autophagosomes, which subsequently fuse with lysosomes. Depending on the involvement of specific receptor proteins, autophagy occurs in a selective or nonselective manner. While this process is well understood at the level of bulky cargo such as mitochondria and bacteria, we know very little about individual proteins and protein complexes that are engulfed and degraded by autophagy. In contrast to the critical role of autophagy in balancing proteostasis, our current knowledge of the autophagic degradome is very limited. Here, we combined proximity labeling with quantitative proteomics to systematically map the protein inventory of autophagosomes. Using this strategy, we uncovered a basal, housekeeping mitophagy pathway that involves piecemeal degradation of mitochondrial proteins in a LC3C- and p62-dependent manner and contributes to mitochondrial homeostasis maintenance when cells rely on oxidative phosphorylation.

Funding information:
  • NIDA NIH HHS - K02 DA026990(United States)

Neuroplastin and Basigin Are Essential Auxiliary Subunits of Plasma Membrane Ca2+-ATPases and Key Regulators of Ca2+ Clearance.

  • Schmidt N
  • Neuron
  • 2017 Nov 15

Literature context: lecular Probes Cat#A31573; RRID:AB_2536183 Anti-mouse-Alexa488, donkey Mol


Abstract:

Plasma membrane Ca2+-ATPases (PMCAs), a family of P-type ATPases, extrude Ca2+ ions from the cytosol to the extracellular space and are considered to be key regulators of Ca2+ signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca2+ transport, as well as submembraneous Ca2+ concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca2+ concentration.

Chromatin and Single-Cell RNA-Seq Profiling Reveal Dynamic Signaling and Metabolic Transitions during Human Spermatogonial Stem Cell Development.

  • Guo J
  • Cell Stem Cell
  • 2017 Oct 5

Literature context: Fisher Scientific cat# A31573, RRID:AB_2536183 Biological Samples


Abstract:

Human adult spermatogonial stem cells (hSSCs) must balance self-renewal and differentiation. To understand how this is achieved, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4+ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4+ hSSCs and differentiating c-KIT+ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic "poising" in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation.

Generation of a human control PBMC derived iPS cell line TUSMi001-A from a healthy male donor of Han Chinese genetic background.

  • Wang Y
  • Stem Cell Res
  • 2017 Oct 17

Literature context: Fisher Scientific Cat# A-31573, RRID:AB_2536183


Abstract:

A 59-year old healthy man of Han Chinese genetic background donated his peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system was used to reprogram his PBMCs with the human OSKM (Oct4, Sox2, Kl4 and c-Myc) transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. In the studies of disease mechanism, the iPSC line can be used as a control.

CTLA-4+PD-1- Memory CD4+ T Cells Critically Contribute to Viral Persistence in Antiretroviral Therapy-Suppressed, SIV-Infected Rhesus Macaques.

  • McGary CS
  • Immunity
  • 2017 Oct 17

Literature context: 7 Invitrogen Cat # A-31573; RRID:AB_2536183 anti-pSTAT5 (clone 47/Stat5(pY6


Abstract:

Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD-1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART-treated, SIV-infected rhesus macaques, we show that CTLA-4+PD-1- memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication-competent and infectious virus. In contrast to PD-1+ Tfh cells, SIV-enriched CTLA-4+PD-1- CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART-mediated viral suppression. We have shown that CTLA-4+PD-1- memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV-1 cure.

Funding information:
  • CCR NIH HHS - HHSN261200800001C()
  • NCI NIH HHS - HHSN261200800001E()
  • NHGRI NIH HHS - P01 HG004120(United States)
  • NIAID NIH HHS - P30 AI050409()
  • NIAID NIH HHS - R01 AI110334()
  • NIAID NIH HHS - R01 AI116379()
  • NIAID NIH HHS - R33 AI104278()
  • NIAID NIH HHS - R33 AI116171()
  • NIH HHS - P51 OD011132()

Aldosterone-Sensing Neurons in the NTS Exhibit State-Dependent Pacemaker Activity and Drive Sodium Appetite via Synergy with Angiotensin II Signaling.

  • Resch JM
  • Neuron
  • 2017 Sep 27

Literature context: gate Invitrogen Cat #: A-31573; RRID:AB_2536183 Chemicals, Peptides, and Recomb


Abstract:

Sodium deficiency increases angiotensin II (ATII) and aldosterone, which synergistically stimulate sodium retention and consumption. Recently, ATII-responsive neurons in the subfornical organ (SFO) and aldosterone-sensitive neurons in the nucleus of the solitary tract (NTSHSD2 neurons) were shown to drive sodium appetite. Here we investigate the basis for NTSHSD2 neuron activation, identify the circuit by which NTSHSD2 neurons drive appetite, and uncover an interaction between the NTSHSD2 circuit and ATII signaling. NTSHSD2 neurons respond to sodium deficiency with spontaneous pacemaker-like activity-the consequence of "cardiac" HCN and Nav1.5 channels. Remarkably, NTSHSD2 neurons are necessary for sodium appetite, and with concurrent ATII signaling their activity is sufficient to produce rapid consumption. Importantly, NTSHSD2 neurons stimulate appetite via projections to the vlBNST, which is also the effector site for ATII-responsive SFO neurons. The interaction between angiotensin signaling and NTSHSD2 neurons provides a neuronal context for the long-standing "synergy hypothesis" of sodium appetite regulation.

Funding information:
  • NIDDK NIH HHS - F32 DK103387()
  • NIDDK NIH HHS - P30 DK046200()
  • NIDDK NIH HHS - P30 DK057521()
  • NIDDK NIH HHS - R01 DK075632()
  • NIDDK NIH HHS - R01 DK089044()
  • NIDDK NIH HHS - R01 DK096010()
  • NIDDK NIH HHS - R01 DK111401()
  • NINDS NIH HHS - K08 NS099425()

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura.

  • Tischfield MA
  • Dev. Cell
  • 2017 Sep 11

Literature context: itrogen (Thermo Fisher)A-21207, A-31573Donkey anti-Goat IgG (H+L) (1:10


Abstract:

Dural cerebral veins (CV) are required for cerebrospinal fluid reabsorption and brain homeostasis, but mechanisms that regulate their growth and remodeling are unknown. We report molecular and cellular processes that regulate dural CV development in mammals and describe venous malformations in humans with craniosynostosis and TWIST1 mutations that are recapitulated in mouse models. Surprisingly, Twist1 is dispensable in endothelial cells but required for specification of osteoprogenitor cells that differentiate into preosteoblasts that produce bone morphogenetic proteins (BMPs). Inactivation of Bmp2 and Bmp4 in preosteoblasts and periosteal dura causes skull and CV malformations, similar to humans harboring TWIST1 mutations. Notably, arterial development appears normal, suggesting that morphogens from the skull and dura establish optimal venous networks independent from arterial influences. Collectively, our work establishes a paradigm whereby CV malformations result from primary or secondary loss of paracrine BMP signaling from preosteoblasts and dura, highlighting unique cellular interactions that influence tissue-specific angiogenesis in mammals.

A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages.

  • Tchieu J
  • Cell Stem Cell
  • 2017 Sep 7

Literature context: ermo Fisher Scientific A-31573, RRID:AB_2536183 AlexaFluor Donkey Anti-Mouse 48


Abstract:

Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFβ pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility of this system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CP specification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.

Funding information:
  • NINDS NIH HHS - R01 NS072381()

Physiological and pathophysiological control of synaptic GluN2B-NMDA receptors by the C-terminal domain of amyloid precursor protein.

  • Pousinha PA
  • Elife
  • 2017 Jul 6

Literature context: onjugated secondary antibodies (RRID:AB_2536183) (Invitrogen, France) for 2 hr


Abstract:

The amyloid precursor protein (APP) harbors physiological roles at synapses and is central to Alzheimer's disease (AD) pathogenesis. Evidence suggests that APP intracellular domain (AICD) could regulate synapse function, but the underlying molecular mechanisms remain unknown. We addressed AICD actions at synapses, per se, combining in vivo AICD expression, ex vivo AICD delivery or APP knock-down by in utero electroporation of shRNAs with whole-cell electrophysiology. We report a critical physiological role of AICD in controlling GluN2B-containing NMDA receptors (NMDARs) at immature excitatory synapses, via a transcription-dependent mechanism. We further show that AICD increase in mature neurons, as reported in AD, alters synaptic NMDAR composition to an immature-like GluN2B-rich profile. This disrupts synaptic signal integration, via over-activation of SK channels, and synapse plasticity, phenotypes rescued by GluN2B antagonism. We provide a new physiological role for AICD, which becomes pathological upon AICD increase in mature neurons. Thus, AICD could contribute to AD synaptic failure.

Differentiation of Human Pluripotent Stem Cells into Colonic Organoids via Transient Activation of BMP Signaling.

  • Múnera JO
  • Cell Stem Cell
  • 2017 Jul 6

Literature context: A-31573, RRID:AB_2536183 Alexafluor


Abstract:

Gastric and small intestinal organoids differentiated from human pluripotent stem cells (hPSCs) have revolutionized the study of gastrointestinal development and disease. Distal gut tissues such as cecum and colon, however, have proved considerably more challenging to derive in vitro. Here we report the differentiation of human colonic organoids (HCOs) from hPSCs. We found that BMP signaling is required to establish a posterior SATB2+ domain in developing and postnatal intestinal epithelium. Brief activation of BMP signaling is sufficient to activate a posterior HOX code and direct hPSC-derived gut tube cultures into HCOs. In vitro, HCOs express colonic markers and contained colon-specific cell populations. Following transplantation into mice, HCOs undergo morphogenesis and maturation to form tissue that exhibits molecular, cellular, and morphologic properties of human colon. Together these data show BMP-dependent patterning of human hindgut into HCOs, which will be valuable for studying diseases including colitis and colon cancer.

Funding information:
  • NIAID NIH HHS - U19 AI116491()
  • NIBIB NIH HHS - U18 EB021780()
  • NIDDK NIH HHS - R01 DK070858()
  • NIDDK NIH HHS - R01 DK092456()
  • NIDDK NIH HHS - R01 DK098350()
  • NIDDK NIH HHS - R01 DK102551()
  • NIDDK NIH HHS - U01 DK103117()

Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression.

  • Knowland D
  • Cell
  • 2017 Jul 13

Literature context: (1:1000)RocheCat#11 867 423 001Donkey anti-rabbit Alexa Fluor 647Life TechnologiesA31573Donkey an


Abstract:

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Funding information:
  • NIMH NIH HHS - R01 MH107742()
  • NIMH NIH HHS - R01 MH108594()

Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses Inflammation.

  • Wlodarska M
  • Cell Host Microbe
  • 2017 Jul 12

Literature context: t#A31573; RRID:AB_2536183 Bacterial


Abstract:

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits.

Funding information:
  • NHLBI NIH HHS - HL28785(United States)

Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids.

  • Nikolić MZ
  • Elife
  • 2017 Jun 30

Literature context: er Scientific A31573 1:2000 RRID:AB_2536183 Goat α-rat 647 Thermo Fisher Sc


Abstract:

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.

Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage.

  • Vivas O
  • Elife
  • 2017 Jun 30

Literature context: so A31573 RRID:AB_2536183), Donkey a


Abstract:

CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near -50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.

Funding information:
  • NHLBI NIH HHS - R01 HL085686()
  • NHLBI NIH HHS - R01 HL085870()
  • NINDS NIH HHS - R37 NS008174()

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

  • van Lessen M
  • Elife
  • 2017 May 12

Literature context: #A31573, RRID:AB_2536183), pHrodo R


Abstract:

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

  • Kaucka M
  • Elife
  • 2017 Apr 17

Literature context: 42672, AB_2536183, RRID:AB_1


Abstract:

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

Funding information:
  • NIBIB NIH HHS - R01 EB014877()

Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells.

  • Jiang T
  • Elife
  • 2017 Mar 7

Literature context: 73 RRID:AB_2536183), Alexa Fl


Abstract:

The asymmetric location of stereociliary bundle (hair bundle) on the apical surface of mechanosensory hair cells (HCs) dictates the direction in which a given HC can respond to cues such as sound, head movements, and water pressure. Notably, vestibular sensory organs of the inner ear, the maculae, exhibit a line of polarity reversal (LPR) across which, hair bundles are polarized in a mirror-image pattern. Similarly, HCs in neuromasts of the zebrafish lateral line system are generated as pairs, and two sibling HCs develop opposite hair bundle orientations. Within these sensory organs, expression of the transcription factor Emx2 is restricted to only one side of the LPR in the maculae or one of the two sibling HCs in neuromasts. Emx2 mediates hair bundle polarity reversal in these restricted subsets of HCs and generates the mirror-image pattern of the sensory organs. Downstream effectors of Emx2 control bundle polarity cell-autonomously via heterotrimeric G proteins.

Immunosuppression via Loss of IL2rγ Enhances Long-Term Functional Integration of hESC-Derived Photoreceptors in the Mouse Retina.

  • Zhu J
  • Cell Stem Cell
  • 2017 Mar 2

Literature context: n A31573; RRID:AB_2536183 Alexa Fluo


Abstract:

Loss of photoreceptors is a common endpoint in degenerative retinal diseases. Human pluripotent stem cells provide a potential source for photoreceptor replacement, but, even in mouse models, the efficiency and efficacy of transplantation-based repair remains poor. In this study, we examined the degree to which immune rejection contributes to these disappointing outcomes using an immunodeficient IL2 receptor γ (IL2rγ)-null mouse model. Our results show that prevention of cell rejection in the normal and degenerating retinal environment significantly improves long-term survival and integration of hESC-derived donor retinal cells. Transplanted cells are able to differentiate into mature photoreceptors expressing various opsins and can functionally integrate into congenitally blind mice. Our work suggests that even though the retina is often considered immune-privileged, suppression of host immune-mediated cell rejection may well be a useful approach for improving long-term integration of transplanted cells with a view to successful clinical outcomes.

Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells.

  • Basak O
  • Cell Stem Cell
  • 2017 Feb 2

Literature context: c A31573, RRID:AB_2536183 AlexaFluor


Abstract:

Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into three-dimensional organoids containing all intestinal epithelial cell types at near-normal ratios. Conditions to generate the main cell types (enterocyte, goblet cells, Paneth cells, and M cells) are well established, but signals to induce the spectrum of hormone-producing enteroendocrine cells (EECs) have remained elusive. Here, we induce Lgr5+ stem cell quiescence in vitro by blocking epidermal growth factor receptor (EGFR) or mitogen-associated protein kinase (MAPK) signaling pathways in organoids and show that their quiescent state is readily reverted. Quiescent Lgr5+ stem cells acquire a distinct molecular signature biased toward EEC differentiation. Indeed, combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates a diversity of EEC hormone-expressing subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and provide an approach for the study of the elusive EECs in a defined environment.

Funding information:
  • NIGMS NIH HHS - T32 GM008061(United States)
  • NINDS NIH HHS - R21NS073585-01A1(United States)

Neonatal disease environment limits the efficacy of retinal transplantation in the LCA8 mouse model.

  • Cho SH
  • BMC Ophthalmol
  • 2016 Nov 4

Literature context: ugated donkey anti-rabbit (Life, {"type":"entrez-protein","attrs":{"text":"A31573","term_id":"87384","term_text":"pir||A31573"}}A31573) antibodies.Go to:ResultsTransp


Abstract:

BACKGROUND: Mutations of Crb1 gene cause irreversible and incurable visual impairment in humans. This study aims to use an LCA8-like mouse model to identify host-mediated responses that might interfere with survival, retinal integration and differentiation of grafted cells during neonatal cell therapy. METHODS: Mixed retinal donor cells (1 ~ 2 × 104) isolated from neural retinas of neonatal eGFP transgenic mice were injected into the subretinal space of LCA8-like model neonatal mice. Markers of specific cell types were used to analyze microglial attraction, CSPG induction and retinal cell differentiation. The positions of host retinal cells were traced according to their laminar location during disease progression to look for host cell rearrangements that might inhibit retinal integration of the transplanted cells. RESULTS: Transplanted retinal cells showed poor survival and attracted microglial cells, but CSPG was not greatly induced. Retinas of the LCA8 model hosts underwent significant cellular rearrangement, including rosette formation and apical displacement of inner retinal cells. CONCLUSIONS: Local disease environment, particularly host immune responses to injected cells and formation of a physical barrier caused by apical migration of host retinal cells upon disruption of outer limiting membrane, may impose two major barriers in LCAs cell transplantation therapy.

Funding information:
  • NINDS NIH HHS - R01 NS083726(United States)

Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts.

  • Loh KH
  • Cell
  • 2016 Aug 25

Literature context: t# A31573 RRID:AB_2536183 Donkey ant


Abstract:

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

  • Pinet-Charvet C
  • Endocrinology
  • 2016 Jan 31

Literature context:


Abstract:

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

Funding information:
  • NIMH NIH HHS - R37 MH063105(United States)

Wild-type neural progenitors divide and differentiate normally in an amyloid-rich environment.

  • Yetman MJ
  • J. Neurosci.
  • 2013 Oct 30

Literature context: ey anti-rabbit IgG (Invitrogen, A-31573). Sections were coverslipped in


Abstract:

Adult neurogenesis is modulated by a balance of extrinsic signals and intrinsic responses that maintain production of new granule cells in the hippocampus. Disorders that disrupt the proliferative niche can impair this process, and alterations in adult neurogenesis have been described in human autopsy tissue and transgenic mouse models of Alzheimer's disease. Because exogenous application of aggregated Aβ peptide is neurotoxic in vitro and extracellular Aβ deposits are the main pathological feature recapitulated by mouse models, cell-extrinsic effects of Aβ accumulation were thought to underlie the breakdown of hippocampal neurogenesis observed in Alzheimer's models. We tested this hypothesis using a bigenic mouse in which transgenic expression of APP was restricted to mature projection neurons. These mice allowed us to examine how wild-type neural progenitor cells responded to high levels of Aβ released from neighboring granule neurons. We find that the proliferation, determination, and survival of hippocampal adult-born granule neurons are unaffected in the APP bigenic mice, despite abundant amyloid pathology and robust neuroinflammation. Our findings suggest that Aβ accumulation is insufficient to impair adult hippocampal neurogenesis, and that factors other than amyloid pathology may account for the neurogenic deficits observed in transgenic models with more widespread APP expression.