identification-analysis-mouse-erythroid-progenitors-using-cd71ter119

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

isolation-quiescent-stem-cell-populations-from-individual-skeletal

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...

identification-analysis-myogenic-progenitors-vivo-during-acute

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle ...

Aarhus University

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. Here we explore cellular and molecular aspects of neural injury signaling in Drosophila. We show that transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wlds protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment.

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons.

Neuron-Glia Kommunikation steht im Mittelpunkt aller Nervensystem Reaktionen auf ein Trauma, dennoch neuronalen Sch&auml;digung signalisieren, dass Wege bleiben schlecht verstanden. Hier erkunden wir zellul&auml;re und Molekulare Aspekte der neuronalen Sch&auml;digung signalisieren in Drosophila. Wir zeigen, dass transected Taufliege Axone Sch&auml;digung induziert Entartung, die morphologisch zu Wallersche Degeneration bei S&auml;ugetieren &auml;hnelt zu unterziehen und werden, indem die neuroprotektive Maus Wlds Protein unterdr&uuml;ckt k&ouml;nnen. Axonale Sch&auml;digung, l&ouml;st starke molekulargenetischer und morphologischer Reaktionen von Drosophila Glia: Glia Upregulate Ausdruck des Rezeptors Engulfment Draper, dramatischen Ver&auml;nderungen der Morphologie und schnell rekrutieren zellul&auml;re Prozesse in Richtung abgetrennten Axone. Draper Mutanten Glia nicht morphologisch Axon Verletzungen zu reagieren und abgetrennten Axone sind nicht gereinigt, aus CNS. So erscheint Draper als ein Gliazellen Rezeptor f&uuml;r abgetrennte Axon abgeleitete Molekulare Signale, dass Laufwerk-Einstellung von Gliazellen Prozesse zu verletzten Axone f&uuml;r Engulfment handeln.

Der Drosophila Zelle Leiche Engulfment Rezeptor Draper vermittelt Gliazellen Abfertigung der abgetrennten Axone.

Tissue development is regulated by signaling networks that control developmental rate and determine ultimate tissue mass. Here we present a novel computational algorithm used to identify regulatory feedback and feedforward interactions between progenitors in developing erythroid tissue. The algorithm makes use of dynamic measurements of red cell progenitors between embryonic days 12 and 15 in the mouse. It selects for intercellular interactions that reproduce the erythroid developmental process and endow it with robustness to external perturbations. This analysis predicts that negative autoregulatory interactions arise between early erythroblasts of similar maturation stage. By studying embryos mutant for the death receptor FAS, or for its ligand, FASL, and by measuring the rate of FAS-mediated apoptosis in vivo, we show that FAS and FASL are pivotal negative regulators of fetal erythropoiesis, in the manner predicted by the computational model. We suggest that apoptosis in erythroid development mediates robust homeostasis regulating the number of red blood cells reaching maturity.

Negative autoregulation by FAS mediates robust fetal erythropoiesis.

Gewebe Entwicklung Signalling Netzwerke, die ultimative Gewebe zu bestimmen und Steuern Entwicklungsst&ouml;rungen Tarif geregelt ist Masse. Hier pr&auml;sentieren wir einen neuartigen computational Algorithmus zur Identifizierung von regulatorischen Feedback und Feedforward Interaktionen zwischen Vorl&auml;uferzellen Erythroid Gewebe zu entwickeln. Der Algorithmus nutzt dynamische Messungen der roten Vorl&auml;uferzellen zwischen embryonalen Tagen 12 und 15 in der Maus. Es w&auml;hlt f&uuml;r interzellul&auml;re Wechselwirkungen, die reproduzieren des Entwicklungsprozesses Erythroid und statte es mit Widerstandsf&auml;higkeit gegen&uuml;ber externen St&ouml;rungen. Diese Analyse prognostiziert, dass negative Autoregulatory Interaktionen zwischen fr&uuml;hen Erythroblasten &auml;hnliche Phase der Reifung entstehen. Durch die Untersuchung von Embryonen Mutant f&uuml;r den Tod-Rezeptor FAS oder f&uuml;r seine Ligand, FASL, und durch die Messrate von FAS-vermittelter Apoptose in-vivo zeigen wir, dass FAS und FASL zentrale negative Regler der fetalen Erythropoese, in der Weise der computational Modell vorhergesagt werden. Wir schlagen vor, dass Apoptosis in Erythroid vermittelt Entwicklung robuster Hom&ouml;ostase Regelung die Anzahl der roten Blutk&ouml;rperchen erreichen der Geschlechtsreife.

Negative Selbstregelung von FAS vermittelt robuste fetalen Erythropoese.

In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild-type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-alpha. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.

Lymphocytic choriomeningitis virus (LCMV) infection of CNS glial cells results in TLR2-MyD88/Mal-dependent inflammatory responses.

Als Reaktion auf eindringende Krankheitserreger spielen Toll-Like Rezeptoren (TLR) eine entscheidende Rolle in der Einleitung der angeborenen Immunantwort, der entweder vorteilhaft oder nachteilig auf den Host sein. In der vorliegenden Studie zeigten wir, dass Gliazellen zentrales Nervensystem (ZNS) durch eine lymphozyt&auml;re Choriomeningitis-Virus (LCM) TLR2-MyD88/Mal-abh&auml;ngigen Weise aktiviert werden. Insbesondere als Reaktion auf LCM isoliert, Astrozyten und Microglial Zellen aus Wildtyp (WT) M&auml;use produzierte Chemokine, wie MCP-1, RANTES und TNF-Alpha. &Auml;hnliche Reaktionen traten in KO TLR3 und TLR4 KO Gliazellen. In auff&auml;lligem Kontrast, Astrozyten und Microglial Zellen von M&auml;usen, die einen Mangel an TLR2, MyD88, isoliert und Mal keiner diese Chemokine produzieren. Dar&uuml;ber hinaus induzierte LCM Infektion von Gliazellen bis-Regulierung von TLR2, MHC-Klasse-I und II, CD40, CD86 MyD88-abh&auml;ngigen Weise. Diese Ergebnisse definieren eine funktionale Rolle f&uuml;r TLR-Signalisierung in virale Infektion-induzierte Aktivierung des CNS Gliazellen sowie f&uuml;r die Immunpathologie im ZNS.

Lymphozyt&auml;re Choriomeningitis-Virus (LCM)-Infektion der CNS Gliazellen f&uuml;hrt TLR2-MyD88/Mal-abh&auml;ngigen entz&uuml;ndliche Reaktionen.

Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, reproduction and aging in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the induction of antimicrobial peptide (AMP) genes in Drosophila melanogaster. 20E pretreatment of Schneider S2 cells promoted the robust induction of AMP genes, following immune stimulation. On the other hand, JH III, and its synthetic analogs (JHa) methoprene and pyriproxyfen, strongly interfered with this 20E-dependent immune potentiation, although these hormones did not inhibit other 20E-induced cellular changes. Similarly, in vivo analyses in adult flies confirmed that JH is a hormonal immuno-suppressor. RNA silencing of either partner of the ecdysone receptor heterodimer (EcR or Usp) in S2 cells prevented the 20E-induced immune potentiation. In contrast, silencing methoprene-tolerant (Met), a candidate JH receptor, did not impair immuno-suppression by JH III and JHa, indicating that in this context MET is not a necessary JH receptor. Our results suggest that 20E and JH play major roles in the regulation of gene expression in response to immune challenge.

Hormonal regulation of the humoral innate immune response in Drosophila melanogaster.

Juvenilhormon (JH) und 20-hydroxy-Ecdyson (20E) sind sehr vielseitig Hormone, koordinierende Entwicklung, Wachstum, Reproduktion und Alterung bei Insekten. Impulse von 20E geben wichtige Signale f&uuml;r die Einleitung von Entwicklungs- und physiologischen &Uuml;berg&auml;nge, w&auml;hrend JH f&ouml;rdert oder diese Signale auf eine B&uuml;hne-spezifische Weise hemmt. Vorherige Anhaltspunkte daf&uuml;r, dass JH und 20E m&ouml;glicherweise Angeborene Immunit&auml;t modulieren, aber es unklar, ob und wie diese Hormone interagieren regulieren die Immunantwort bleibt. Hier zeigen wir, dass JH und 20E antagonistische Wirkungen auf die Induktion von Genen, antimikrobielle Peptide (AMP) in Drosophila Melanogaster. 20E Vorbehandlung der Schneider S2-Zellen bef&ouml;rdert die robuste Induktion AMP Gene, folgenden immun-Stimulierung. Auf der anderen Seite behinderten JH III, und seine synthetischen Analoga (JI)-Methopren und Pyriproxyfen, stark diese 20E-abh&auml;ngige immun Potenzierung, obwohl diese Hormone nicht andere 20E-induzierte zellul&auml;ren Ver&auml;nderungen hemmen. Ebenso best&auml;tigt in-vivo-Analysen in Erwachsenen fliegen, dass JH eine hormonelle Immuno-Schutzbeschaltung. RNA silencing der beiden Partner die Ecdyson-Rezeptor-Heterodimer (EcR oder Usp) in S2-Zellen verhindert die 20E-induzierte immun Potenzierung. Dagegen Unterdr&uuml;ckender Methopren-tolerant (MWB), ein Kandidat-JH-Rezeptor, Immuno-Unterdr&uuml;ckung von JH III nicht beeintr&auml;chtigen und JHa, zeigt an, dass in diesem Zusammenhang MET ist kein erforderlichen JH-Rezeptor. Unsere Ergebnisse legen nahe, dass 20E und JH wichtige Rollen bei der Regulierung der Genexpression in Reaktion auf immun-Herausforderung spielen.

Die Hormonale Regelung der angeborene humorale Immunantwort in Drosophila Melanogaster.

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and Î²-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-x(L) is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-x(L) pathway "resets," allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using "knock-in" mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-x(L) response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-x(L) induction provides a "stop-gap" in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-x(L) may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways.

&Uuml;berleben, die Signalisierung durch Erythropoetin (Epo) Rezeptor (EpoR) ist wesentlich f&uuml;r die Erythropoese und seine Beschleunigung Hypoxie Stress. Mehrere scheinbar redundante EpoR &uuml;berleben Bahnen wurden in vitro erh&ouml;hen die M&ouml;glichkeit, ihre funktionale Spezialisierung in-vivo identifiziert. Hier haben wir Mausmodelle von akuter und chronischer Stress, einschlie&szlig;lich einer Hypoxie Umwelt und &beta;-Thalass&auml;mie, verwendet, um zwei deutlich verschiedene Antwort-Dynamik f&uuml;r zwei Erythroblast &uuml;berleben Wege in-vivo zu identifizieren. Induktion des Proteins Antiapoptotic Bcl-x(L) ist rapid aber vor&uuml;bergehend, w&auml;hrend Unterdr&uuml;ckung von Proapoptotischen Protein Bim langsamer, aber beharrlichen. &Auml;hnlich wie sensorische Anpassung, &quot;setzt jedoch die Bcl-x(L)-Pathway,&quot; erm&ouml;glicht es, einen Neuanfang auf akuten Stress &uuml;berlagert ein chronischer Stress-Reiz zu reagieren. Mit &quot;Knock-in&quot; Maus Modelle Mutant EpoRs zum Ausdruck zu bringen, wir fanden, dass Anpassung in der Bcl-x(L)-Antwort aufgrund der Anpassung der seine upstream Regler Stat5 auftritt, erfordern beide die EpoR distale zytoplasmatische Dom&auml;ne. Wir schlie&szlig;en, dass &uuml;berleben Bahnen vorher unvermutetes funktionale Spezialisierung f&uuml;r die akuten und chronischen Phasen der Stress-Reaktion zeigen. BCL-x(L) Induktion bietet eine &quot;Stop-L&uuml;cke&quot; in akutem Stress bis langsamer aber permanente Bahnen werden aktiviert. Dar&uuml;ber hinaus kann pathologische Erh&ouml;hung von Bcl-x(L) das Ergebnis gest&ouml;rter Anpassung, mit Auswirkungen f&uuml;r myeloproliferative Krankheitsmechanismen sein.

Kontrastierende dynamischer Antworten in-vivo der Bcl-xL und Bim erythropoetische &uuml;berleben Bahnen.

Trogocytosis is an active transport mechanism by which one cell extracts a plasma membrane fragment with embedded molecules from an adjacent cell in a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting key experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. For complete details on the use and execution of this protocol, please refer to Gonzalez et al. (2021).

Measuring trogocytosis between ovarian tumor and natural killer cells.

We are living longer, but our healthspan has not increased. The goal of regenerative medicine is to increase quality of life through an understanding of the cellular and molecular processes that underlie effective tissue repair in order to restore damaged tissues. The drivers of muscle regeneration are the muscle stem cells that cycle between quiescent and activated states to meet tissue regenerative demands. Here we review recent findings on the role of the niche, or tissue microenvironment, in the modulation of muscle stem cell plasticity and the mechanisms responsible for the drastic loss of stem cell function with aging. These new studies unveil fundamental mechanisms of stem cell plasticity with broad relevance to other tissues and lay the foundation for the development of therapeutic strategies to boost the regenerative potential of aged muscle stem cells.

Plasticity of muscle stem cells in homeostasis and aging.

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47) with impaired regenerative capacity that predominate with aging. The prevalent CD47 MuSC subset suppresses the residual functional CD47 MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

ArticleTotal : 3	Year
Publication title Cited by 147	2011
Publication title Cited by 4	2022
Publication title Cited by 2	2023

Article	Year
The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron\| PubMed ID: 16772169	2006
Negative autoregulation by FAS mediates robust fetal erythropoiesis. PLoS biology\| PubMed ID: 17896863	2007
Lymphocytic choriomeningitis virus (LCMV) infection of CNS glial cells results in TLR2-MyD88/Mal-dependent inflammatory responses. Journal of neuroimmunology\| PubMed ID: 18295350	2008
Hormonal regulation of the humoral innate immune response in Drosophila melanogaster. The Journal of experimental biology\| PubMed ID: 18689425	2008
Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways. Blood\| PubMed ID: 22086418	2012
Measuring trogocytosis between ovarian tumor and natural killer cells. STAR protocols\| PubMed ID: 35693208	2022
Plasticity of muscle stem cells in homeostasis and aging. Current opinion in genetics & development\| PubMed ID: 36308777	2022
Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration. Cell stem cell\| PubMed ID: 36384141	2022
Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance. Science advances\| PubMed ID: 36753540	2023

Ermelinda Porpiglia

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