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 ...

The purpose of this study was to report the angiographic findings of the first human evaluation of the everolimus-eluting stent (EES) for the treatment of noncomplex coronary lesions. Forty-two patients with de novo coronary lesions (2.75 to 4.00 mm vessels; lesion length, <18 mm) were prospectively randomized in a 2:1 ratio to receive either the EES (n = 27) or a metallic stent (n = 15). Baseline clinical and angiographic characteristics were similar among both groups. At 6-month follow-up, EES had a lower in-stent late lumen loss (0.10 +/- 0.22 vs 0.85 +/- 0.32 mm, p <0.0001) and in-segment diameter stenoses (20.7 +/- 12.3% vs 37.0 +/- 15.8%, p = 0.002). There was no in-stent restenosis with EES; however, 1 focal distal edge restenosis was present. There was 1 in-stent and 1 in-segment (proximal edge) restenosis in the metallic stent group. There was no stent thrombosis or aneurysm formation at follow-up in either group.

Angiographic results of the first human experience with everolimus-eluting stents for the treatment of coronary lesions (the FUTURE I trial).

Die angiographischen Ergebnisse der ersten menschlichen Erfahrung mit Everolimus-freisetzenden Stents f&#xFC;r die Behandlung von koronaren L&#xE4;sionen (die Zukunft I-Studie).

Previously, our group reported a five-generation family in which a balanced t(13;17) translocation is associated with a spectrum of skeletal abnormalities, including Robin sequence, hypoplastic scapulae, and a missing pair of ribs. Using polymerase chain reaction (PCR) with chromosome-specific markers to analyze DBA from somatic cell hybrids containing the derivative translocation chromosomes, we narrowed the breakpoint on each chromosome. Subsequent sequencing of PCR products spanning the breakpoints identified the breaks precisely. The chromosome 17 breakpoint maps approximately 932 kb upstream of the sex-determining region Y (SRY)-related high-mobility group box gene (SOX) within a noncoding transcript represented by two IMAGE cDNA clones. A growing number of reports have implicated chromosome 17 breakpoints at a distance of up to 1 Mb from SOX9 in some cases of campomelic dysplasia (CD). Although this multigeneration family has a disorder that shares some features with CD, their phenotype is significantly milder than any reported cases of (nonmosaic) CD. Therefore, this case may represent an etiologically distinct skeletal dysplasia or may be an extremely mild familial example of CD, caused by the most proximal translocation breakpoint from SOX9 reported to date. In addition, we have refined the breakpoint in a acampomelic CD case described elsewhere and have found that it lies approximately 900 kb upstream of SOX9.

Fine mapping of chromosome 17 translocation breakpoints > or = 900 Kb upstream of SOX9 in acampomelic campomelic dysplasia and a mild, familial skeletal dysplasia.

Fein-Kartierung des Chromosoms 17 Translokation Haltepunkte&#x26;gt; oder = 900 kb stromaufw&#xE4;rts SOX9 in acampomelic campomelic Dysplasie und einem milden, famili&#xE4;re Skelettdysplasie.

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion.

Eine homozygote Nonsense-Mutation in SOX9 in der herrschenden Unordnung campomelic Dysplasie: ein Fall, der mitotischen Genkonversion.

Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highFSChigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and beta-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo.

Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo.

Erythropoetin (Epo) ist der wichtigste Regulator der erythropoetischen Reaktion auf Hypoxie Spannung, durch seinen Rezeptor, EpoR. Die Vermittlung der Stressantwort EpoR-Signale sind weitgehend unbekannt, und das Spektrum der Vorl&auml;uferzellen, die Spannung reagieren ist nicht vollst&auml;ndig definiert. Hier haben wir Durchflusszytometrie verwendet, um Stress-responsive Ter119 + CD71highFSChigh fr&uuml;he Erythroblast Teilmengen in-vivo zu identifizieren. In der Maus Milz, erythropoetische Reservat Organ fr&uuml;he Erythroblasten waren anwesend bei niedrigeren Frequenzen und h&ouml;here Raten von Apoptosis als gleichwertige Zellen im Knochenmark unterzogen wurden. Ein hoher Anteil der Milz fr&uuml;hen Erythroblasten coexpressed den Tod-Rezeptor Fas und sein Ligand, FasL. FAS-positivem fr&uuml;he Erythroblasten waren deutlich h&auml;ufiger zu coexpress annexin V als gleichwertige, Fas-Negative Zellen, was darauf hindeutet, dass Fas vermittelt fr&uuml;hen Erythroblast Apoptosis in-vivo. Wir untersuchten verschiedene Mausmodelle erythropoetische Stress, einschlie&szlig;lich Erythrozytose und Beta-Thalass&auml;mie. Wir fanden einen dramatischen Anstieg der H&auml;ufigkeit der Milz fr&uuml;hen Erythroblasten, die korreliert mit Herunterregulation Fas und FasL aus ihrer Zellenoberfl&auml;che. Dar&uuml;ber hinaus unterdr&uuml;ckt eine einzige Injektion von Epo speziell fr&uuml;he Erythroblast Fas und FasL mRNA und Zelloberfl&auml;che Ausdruck. Daher sind Fas und FasL negativen Regulatoren der Erythropoese. EPO-vermittelte Unterdr&uuml;ckung Erythroblast Fas und FasL ist eine neuartige Spannung Antwort, die Erythroblast in-vivo Erweiterungsm&ouml;glichkeiten.

Unterdr&uuml;ckung der Fas-FasL Coexpression von Erythropoetin vermittelt Erythroblast Expansion in die erythropoetische Stressantwort in-vivo.

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.

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the Î²-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57(KIP2) and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a "synchromesh" mechanism that "locks" the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.

A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.

H&auml;matopoetische Vorl&auml;uferzellen unterziehen Differenzierung w&auml;hrend der Navigation mehrere Zellteilung-Zyklen, aber es ist nicht bekannt, ob diese beiden Prozesse gekoppelt sind. Wir widmeten sich dieser Frage durch das Studium der Erythropoese in Maus fetalen Leber in-vivo. Wir fanden, dass die anf&auml;ngliche Hochregulation der Zelloberfl&auml;che, die entwicklungspolitisch CD71 identifiziert Erythroblasten zugeordnet, die in S-Phase eng synchronisiert werden. Wir zeigen, dass die DNA-Replikation innerhalb dieses aber nicht nachfolgende Zyklen wird ben&ouml;tigt f&uuml;r eine Differenzierung-Schalter, bestehend aus schnellen und gleichzeitige Einlieferung &Uuml;berg&auml;nge deren zeitgenaues zuvor unbekannt war. Dazu geh&ouml;ren den Beginn der Erythropoetin-Abh&auml;ngigkeit, Aktivierung der Erythroid Meister transcriptional Regulator GATA-1 und eine Umstellung auf eine aktive Chromatin-Konformation, bei der &beta;-Globin Locus. Insbesondere Fortschreiten der S-Phase ist erforderlich f&uuml;r die Bildung von DNase I &uuml;berempfindlich Websites und f&uuml;r DNA-Demethylierung am dieses Locus. Mechanistisch, zeigen wir, dass die S-Phase Progression w&auml;hrend dieses Schritts wichtige Zwangseinweisung ist abh&auml;ngig von Heraufregulation des cyclin-abh&auml;ngige Kinase-p57(KIP2) und wiederum f&uuml;hrt die Heraufregulation des PU.1, ein Antagonist des GATA-1-Funktion. Diese Ergebnisse unterstreichen daher eine neue Rolle f&uuml;r ein cyclin-abh&auml;ngige Kinasehemmer bei Differenzierung, unterschiedliche an ihre bekannten Funktion im Zellzyklus-Exit. Dar&uuml;ber hinaus zeigen wir, dass einen Roman, gegenseitige Hemmung zwischen PU.1 Ausdruck und Fortschreiten der S-Phase bietet einen &quot;vollsynchronisiertes&quot; Mechanismus, der das Programm Erythroid Differenzierung der Zellzyklus-Uhr, eine genaue Koordinierung der kritische Differenzierung Ereignisse &quot;aufh&auml;ngt&quot;.

Ein wichtige Verpflichtung Schritt bei der Erythropoese wird mit der Uhr des Zellzyklus durch gegenseitige Hemmung zwischen PU.1 und S-Phasen Fortschreiten synchronisiert.

Survivin is a multifunctional protein with essential roles in cell division and inhibition of apoptosis, but the molecular underpinnings of its cytoprotective properties are poorly understood. Here we show that homozygous deletion of the aryl hydrocarbon receptor-interacting protein (AIP), a survivin-associated immunophilin, causes embryonic lethality in mice by embryonic day 13.5-14, increased apoptosis of Ter119(-)/CD71(-) early erythropoietic progenitors, and loss of survivin expression in its cytosolic and mitochondrial compartments in vivo. In import assays using recombinant proteins, AIP directly mediated the import of survivin to mitochondria, thus enabling its anti-apoptotic function, whereas a survivin 1-141 mutant that does not bind AIP was not imported to mitochondria and failed to inhibit apoptosis. AIP-directed mitochondrial import of survivin did not affect cell division, was independent of the organelle transmembrane potential, did not require the chaperone Heat Shock Protein 90 (Hsp90), and was inhibited by cytosolic factor(s) present in normal cells. shRNA knockdown of the mitochondrial import receptor Tom20 abolished mitochondrial import of survivin and sensitized tumor cells to apoptosis, whereas silencing of Tom70 had no effect. Therefore, an AIP-Tom20 recognition contributes to cell survival in development and cancer by mediating the mitochondrial import of survivin.

Developmental control of apoptosis by the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) involves mitochondrial import of the survivin protein.

Survivin ist ein multifunktionales Protein mit entscheidende Rolle in der Zellteilung und Hemmung der Apoptose, aber die molekularen Grundlagen der Cytoprotective Eigenschaften werden oft missverstanden. Hier zeigen wir, dass homozygote L&ouml;schung von Aryl-Kohlenwasserstoff-Rezeptor-Interaktion Protein (AIP), ein survivin-assoziierten Immunophilin verursacht embryonalen T&ouml;dlichkeit bei M&auml;usen von embryonalen Tag 13,5-14, erh&ouml;hte Apoptose der Ter119(-)/CD71(-) fr&uuml;hen erythropoetischen Vorl&auml;uferzellen und Verlust von survivin Ausdruck in seiner Zytosolische und mitochondriale F&auml;cher, die in-vivo. Import-Assays mit rekombinante Proteinen vermittelte AIP direkt den Import von survivin, Mitochondrien, wodurch seine Anti-Apoptotic Funktion, w&auml;hrend ein survivin 1-141-Mutant, der AIP nicht bindet war nicht in Mitochondrien importiert und nicht die Apoptose zu hemmen. Unter der Regie von AIP mitochondrial Import von survivin hatte keine Auswirkungen auf die Zellteilung, war unabh&auml;ngig von der Organell-Transmembran-Potenzial, das Chaperone Heat Shock Protein 90 (Hsp90) nicht erforderlich und wurde durch Zytosolische Faktor(en) in normalen Zellen gehemmt. ShRNA Niederschlag des Rezeptors mitochondrial Import Tom20 abgeschafft mitochondrial Import von survivin und sensibilisierten Tumorzellen zum Apoptosis, w&auml;hrend zum Schweigen von Tom70 keine Auswirkungen hatte. Daher tr&auml;gt eine AIP-Tom20 Anerkennung zur Zelle &Uuml;berleben in Entwicklung und Krebs durch Vermittlung den mitochondrialen Import von survivin.

Entwicklungsbiologie Kontrolle von Apoptosis durch das Immunophilin Aryl Kohlenwasserstoff-Rezeptor-Interaktion Protein (AIP) bringt mitochondrial Import von survivin Proteins.

In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific Î²-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.

Global DNA demethylation during mouse erythropoiesis in vivo.

Im S&auml;ugetier-Genom 5&#39;-CpG-3&#39; Dinucleotides sind h&auml;ufig methyliert, Korrelation mit transkriptionelle zum Schweigen. Genomweiten Demethylierung ist vermutlich nur zweimal w&auml;hrend der Entwicklung in primordialen Keimzellen und der Pr&auml;implantations Embryo auftreten. Diese Demethylierung Ereignisse folgen de Novo Methylierung, ein Muster in der gesamten Entwicklung geerbt und aktualisiert nur am Gewebe-spezifischen Loci einrichten. Wir studierten die DNA-Methylierung in Maus Erythroblasten in-vivo mithilfe von genomischen operierenden reduzierte Darstellung Bisulfit Sequenzierung (RRBS) zu unterscheiden. Demethylierung bei der Erythroid-spezifische &beta;-Globin Locus wurde mit globalen DNA-Demethylierung am meisten genomische Elemente zusammen. Globale Demethylierung wurde kontinuierlich im gesamten Differenzierung und erforderliche schnelle DNA-Replikation. Daher kann die DNA-Demethylierung Global w&auml;hrend somatische Differenzierung, die ein experimentelles Modell f&uuml;r seine Studie in Entwicklung und Krankheit auftreten.

Article	Year
Angiographic results of the first human experience with everolimus-eluting stents for the treatment of coronary lesions (the FUTURE I trial). The American journal of cardiology\| PubMed ID: 15619406	2005
Fine mapping of chromosome 17 translocation breakpoints > or = 900 Kb upstream of SOX9 in acampomelic campomelic dysplasia and a mild, familial skeletal dysplasia. American journal of human genetics\| PubMed ID: 15717285	2005
A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion. Human genetics\| PubMed ID: 15806394	2005
Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood\| PubMed ID: 16527892	2006
Negative autoregulation by FAS mediates robust fetal erythropoiesis. PLoS biology\| PubMed ID: 17896863	2007
A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS biology\| PubMed ID: 20877475	2010
Developmental control of apoptosis by the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) involves mitochondrial import of the survivin protein. The Journal of biological chemistry\| PubMed ID: 21454573	2011
Global DNA demethylation during mouse erythropoiesis in vivo. Science (New York, N.Y.)\| PubMed ID: 22076376	2011

Ramona Pop

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