Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

University of Freiburg

&#x524D;&#x8111;&#xFF0C;&#x8868;&#x8FBE;&#x8F6C;&#x5316;&#x751F;&#x957F;&#x56E0;&#x5B50; &beta; (Tgfbeta) &#x548C;&#x76F8;&#x5173;&#x4FE1;&#x53F7;&#x4EA7;&#x751F;&#x5F71;&#x54CD;&#x7684;&#x624B;&#x6BB5;&#xFF0C;&#x4F46;&#x4E86;&#x89E3;&#x751A;&#x5C11;&#x7684;&#x8FD9;&#x79CD;&#x591A;&#x529F;&#x80FD;&#x7684;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#x4F5C;&#x7528;&#x524D;&#x8111;&#x53D1;&#x80B2;&#x8FC7;&#x7A0B;&#x3002;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x4F7F;&#x7528;&#x7684;&#x53D1;&#x5C55;&#x4E2D;&#x56FD;&#x5BB6;&#x8001;&#x9F20;&#x7684;&#x5927;&#x8111;&#x6D77;&#x9A6C;&#x548C;&#x76AE;&#x5C42;&#x4E3B;&#x7EC6;&#x80DE;&#x57F9;&#x517B;&#xFF0C;&#x53D1;&#x73B0; Tgfbeta &#x57FA;&#x56E0;&#x4E0D;&#x4EC5;&#x5173;&#x8054;&#x4E0E;&#x7956;&#x7EC6;&#x80DE;&#x7684;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x9000;&#x51FA;&#x4F46;&#x4E5F;&#x901A;&#x8FC7;&#x4E86;&#x795E;&#x7ECF;&#x5143;&#x7EC6;&#x80DE;&#x547D;&#x8FD0;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x8BA4;&#x4E3A;&#x4E0D;&#x4EC5; Tgfbeta antimitotic &#x5BF9;&#x7956;&#x7EC6;&#x80DE;&#x4E5F;&#x589E;&#x52A0;&#x7684;&#x8868;&#x8FBE;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x6807;&#x5FD7;&#x7269;&#x6CBB;&#x7597; Tgfbeta &#x6587;&#x5316;&#x3002;&#x8FD9;&#x79CD;&#x6548;&#x679C;&#x662F;&#x53D6;&#x51B3;&#x4E8E; Smad4&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x4F7F;&#x7528; Tgfbeta2(-/-)/Tgfbeta3(-/-) &#x53CC;&#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5C0F;&#x9F20;&#x4F53;&#x5185;&#x635F;&#x5931;&#x7684;&#x51FD;&#x6570;&#x5206;&#x6790;&#x8868;&#x660E;&#x76F8;&#x53CD;&#x589E;&#x52A0;&#x7684;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x7684;&#x6548;&#x679C;&#x548C;&#x66F4;&#x5C11;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x5728;&#x5927;&#x8111;&#x76AE;&#x8D28;&#x548C;&#x6D77;&#x9A6C;&#x3002;&#x53D7; Tgfbeta &#x548C;&#x5DF2;&#x77E5;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x7956;&#x7EC6;&#x80DE;&#x7684;&#x53D1;&#x80B2;&#x8FC7;&#x7A0B;&#x4E2D;&#x6240;&#x6D89;&#x53CA;&#x7684;&#x5019;&#x9009;&#x57FA;&#x56E0; Gata2&#x3001; Runx1 &#x548C; Nedd9&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528; siRNA &#x4ECB;&#x5BFC;&#x7684;&#x7EC4;&#x5408;&#x5F0F;&#xFF0C;&#x4F5C;&#x4E3A; Tgfbeta &#x4F9D;&#x8D56;&#x589E;&#x52A0;&#x795E;&#x7ECF;&#x5143;&#x7EC6;&#x80DE;&#x547D;&#x8FD0;&#x7684;&#x91CD;&#x8981;&#x7EC4;&#x6210;&#x90E8;&#x5206;&#x4FE1;&#x53F7;&#x786E;&#x5B9A; Nedd9&#x3002;&#x6B64;&#x811A;&#x624B;&#x67B6;&#x86CB;&#x767D;&#xFF0C;&#x8FD9;&#x662F;&#x4E3B;&#x8981;&#x63CF;&#x8FF0;&#x4E3A; beta1 &#x6574;&#x5408;&#x901A;&#x8DEF;&#x4FE1;&#x53F7;&#x5206;&#x5B50;&#xFF0C;&#x8868;&#x8FBE;&#x7684;&#x662F;&#x4E0D;&#x53EA;&#x8BF1;&#x5BFC;&#x540E;&#xFF0C;&#x800C;&#x4E14; Tgfbeta &#x6CBB;&#x7597;&#x4E5F;&#x662F;&#x4E00;&#x4E2A;&#x4E0E;&#x5F62;&#x6001;&#x5B66;&#x6539;&#x53D8;&#x7684;&#x89C2;&#x5BDF;&#x5230;&#x56E0;&#x66B4;&#x9732;&#x4E8E; Tgfbeta &#x7684;&#x5DE2;&#x86CB;&#x767D;&#x9633;&#x6027;&#x7956;&#x6C60;&#x76F8;&#x5173;&#x8054;&#x3002;

&#x8F6C;&#x5316;&#x751F;&#x957F;&#x56E0;&#x5B50; &beta; &#x4FC3;&#x8FDB;&#x795E;&#x7ECF;&#x7EC6;&#x80DE;&#x7684;&#x9F20;&#x6807;&#x76AE;&#x8D28;&#x548C;&#x6D77;&#x9A6C;&#x7956;&#x7EC6;&#x80DE;&#x4F53;&#x5916;&#x548C;&#x4F53;&#x5185;&#x7684;&#x547D;&#x8FD0;&#xFF1A; &#x9274;&#x5B9A; Nedd9 &#x4F5C;&#x4E3A;&#x4FE1;&#x53F7;&#x4F20;&#x8F93;&#x7684;&#x91CD;&#x8981;&#x7EC4;&#x6210;&#x90E8;&#x5206;&#x3002;

Transforming Growth Factor beta (Tgfbeta) and associated signaling effectors are expressed in the forebrain, but little is known about the role of this multifunctional cytokine during forebrain development. Using hippocampal and cortical primary cell cultures of developing mouse brains, this study identified Tgfbeta-regulated genes not only associated with cell cycle exit of progenitors but also with adoption of neuronal cell fate. Accordingly, we observed not only an antimitotic effect of Tgfbeta on progenitors but also an increased expression of neuronal markers in Tgfbeta treated cultures. This effect was dependent upon Smad4. Furthermore, in vivo loss-of-function analyses using Tgfbeta2(-/-)/Tgfbeta3(-/-) double mutant mice showed the opposite effect of increased cell proliferation and fewer neurons in the cerebral cortex and hippocampus. Gata2, Runx1, and Nedd9 were candidate genes regulated by Tgfbeta and known to be involved in developmental processes of neuronal progenitors. Using siRNA-mediated knockdown, we identified Nedd9 as an essential signaling component for the Tgfbeta-dependent increase in neuronal cell fate. Expression of this scaffolding protein, which is mainly described as a signaling molecule of the beta1-integrin pathway, was not only induced after Tgfbeta treatment but was also associated with morphological changes of the Nestin-positive progenitor pool observed upon exposure to Tgfbeta.

Transforming growth factor beta promotes neuronal cell fate of mouse cortical and hippocampal progenitors in vitro and in vivo: identification of Nedd9 as an essential signaling component.

Transforming Growth Factor beta (TGF) et effecteurs signalisation associ&eacute;es sont exprim&eacute;s dans le cerveau ant&eacute;rieur, mais on conna&icirc;t mal le r&ocirc;le de cette cytokine multifonctionnelle au cours du d&eacute;veloppement du cerveau. En utilisant des cultures de cellules primaires de hippocampe et cortex de d&eacute;velopper des cerveaux de souris, cette &eacute;tude a identifi&eacute; des g&egrave;nes r&eacute;gul&eacute;s par le TGF associ&eacute;s non seulement avec la sortie du cycle cellulaire des cellules souches, mais aussi avec l&#39;adoption du destin des cellules neuronales. En cons&eacute;quence, nous avons observ&eacute; non seulement un effet antimitotique de TGF sur les cellules souches, mais aussi une augmentation de l&#39;expression de marqueurs neurones dans les cultures trait&eacute;e de TGF. Cet effet d&eacute;pendait de Smad4. En outre, les analyses de perte de fonction in vivo chez des souris mutantes double Tgfbeta2(-/-)/Tgfbeta3(-/-) a montr&eacute; l&#39;effet inverse de la prolif&eacute;ration cellulaire accrue et moins de neurones dans le cortex c&eacute;r&eacute;bral et l&#39;hippocampe. Gata2, Runx1 et Nedd9 &eacute;taient des g&egrave;nes candidats r&eacute;gi par TGF et connus pour &ecirc;tre impliqu&eacute;s dans le processus de d&eacute;veloppement des cellules souches neuronales. &Agrave; l&#39;aide de knockdown m&eacute;di&eacute;e par les siARN, nous avons identifi&eacute; Nedd9 comme une composante essentielle de signalisation pour l&#39;augmentation de TGF-d&eacute;pendante dans le destin des cellules neuronales. Expression de cette prot&eacute;ine d&#39;&eacute;chafaudage, qui est principalement d&eacute;crite comme une mol&eacute;cule de signalisation de la voie de l&#39;int&eacute;grine beta1, n&#39;est pas seulement induite apr&egrave;s que traitement TGF mais &eacute;tait &eacute;galement li&eacute;e &agrave; des modifications morphologiques de la piscine de prog&eacute;niteurs Nestin positif observ&eacute; lors de l&#39;exposition au TGF.

Transforming growth factor beta favorise sort de cellules neuronales des prog&eacute;niteurs de corticale et hippocampe souris in vitro et in vivo : identification des Nedd9 comme une composante essentielle de signalisation.

Transforming-Growth-Faktor-Beta (Tgfbeta) und damit verbundenen Signalling Effektoren in das Stirnhirn ausgedr&uuml;ckt, aber ist wenig bekannt &uuml;ber die Rolle von dieser multifunktionalen Cytokine w&auml;hrend der Entwicklung des Stirnhirns. Mit erh&ouml;hten Cortisolspiegels und kortikale prim&auml;re Zellkulturen Maus Gehirne zu entwickeln, diese Studie identifiziert Tgfbeta-regulierten Genen nicht nur mit Zellzyklus Ausfahrt Vorreiter, aber auch mit Annahme des neuronalen Zelle Schicksal. Dementsprechend beobachteten wir nicht nur die antimitotic Beeinflussung der Tgfbeta Vorl&auml;uferzellen, sondern auch eine erh&ouml;hte Expression neuronaler Marker in Tgfbeta behandelten Kulturen. Dieser Effekt war Smad4 abh&auml;ngig. Ferner zeigte in-vivo Loss of Function-Analysen mit Tgfbeta2(-/-)/Tgfbeta3(-/-) doppelte Mutante M&auml;use den gegenteiligen Effekt des erh&ouml;hten Zellproliferation und weniger Neuronen in der Gro&szlig;hirnrinde und Hippocampus. Gata2, Runx1 und Nedd9 waren Kandidatengene reguliert durch Tgfbeta und bekannt, um Entwicklungsprozesse der neuronalen Vorl&auml;uferzellen beteiligt sein. Mittels SiRNA-vermittelte Niederschlag, identifiziert wir Nedd9 als wesentliche signalisierende Komponente f&uuml;r den Tgfbeta-abh&auml;ngige Anstieg der neuronalen Zelle Schicksal. Ausdruck dieses Ger&uuml;st-Proteins, die vor allem als eine signalisierende Molek&uuml;l von der beta1-Integrin-Weg beschrieben ist, wurde nicht nur ausgel&ouml;st, nachdem Tgfbeta Behandlung aber auch morphologische Ver&auml;nderungen des Nestin-positiven Vorl&auml;uferzellen Pools beobachtet nach Exposition gegen&uuml;ber Tgfbeta zugeordnet war.

Transforming Growth Factor Beta f&ouml;rdert neuronale Zelle Schicksal der Maus kortikale und hippocampal Vorl&auml;uferzellen in vitro und in-vivo: Identifizierung von Nedd9 als wesentliche signalisierende Komponente.

Transforming Growth Factor beta (Tgfbeta) e associati segnalazione effettori sono espressi nel proencefalo, ma poco si sa circa il ruolo di questa citochina multifunzionale durante lo sviluppo del proencefalo. Utilizzando colture cellulari primarie ippocampale e corticale di sviluppo cervello del mouse, questo studio identificato geni regolati Tgfbeta associate non solo con l&#39;uscita del ciclo cellulare dei progenitori ma anche con l&#39;adozione del destino delle cellule neuronali. Di conseguenza, abbiamo osservato non solo un effetto antimitotici Tgfbeta su progenitori, ma anche un&#39;espressione aumentata dei marker neuronale nelle culture Tgfbeta trattato. Questo effetto era dipendente da Smad4. Inoltre, analisi di perdita di funzione in vivo utilizzando topi mutanti doppi Tgfbeta2(-/-)/Tgfbeta3(-/-) ha mostrato l&#39;effetto opposto di aumento della proliferazione cellulare e un minor numero di neuroni nell&#39;ippocampo e corteccia cerebrale. Gata2, Runx1 e Nedd9 erano geni regolati da Tgfbeta e noto per essere coinvolto nei processi di sviluppo dei progenitori neuronali. Utilizzando siRNA-mediata atterramento, abbiamo identificato Nedd9 come una componente essenziale di segnalazione per l&#39;aumento di Tgfbeta-dipendente nel destino delle cellule neuronali. Espressione di questa proteina di ponteggi, che &egrave; principalmente descritta come una molecola di segnalazione del percorso beta1-integrina, non &egrave; stata indotta solo dopo trattamento Tgfbeta ma era anche associato con cambiamenti morfologici della piscina progenitore Nestin-positivi osservati in seguito all&#39;esposizione a Tgfbeta.

Transforming growth factor beta promuove il destino delle cellule neuronali di progenitori corticale e hippocampal del mouse in vitro e in vivo: l&#39;identificazione di Nedd9 come una componente essenziale di segnalazione.

&#x6210;&#x9577;&#x56E0;&#x5B50; &beta; &#xFF08;tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;) &#x3068;&#x95A2;&#x9023;&#x3059;&#x308B;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0; &#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x3001;&#x524D;&#x8133;&#x3067;&#x8868;&#x3055;&#x308C;&#x308B;&#x304C;&#x3001;&#x5C11;&#x3057;&#x524D;&#x8133;&#x958B;&#x767A;&#x4E2D;&#x306B;&#x3053;&#x306E;&#x591A;&#x6A5F;&#x80FD;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x306E;&#x5F79;&#x5272;&#x306B;&#x3064;&#x3044;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x8133;&#x306E;&#x958B;&#x767A;&#x306E;&#x6D77;&#x99AC;&#x3068;&#x5927;&#x8133;&#x76AE;&#x8CEA;&#x306E;&#x4E00;&#x6B21;&#x96FB;&#x6C60;&#x6587;&#x5316;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x3053;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x898F;&#x5236;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x304B;&#x3089;&#x306E;&#x96E2;&#x8131;&#x3082;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x306E;&#x63A1;&#x7528;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x8B58;&#x5225;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x6297;&#x304C;&#x3093;&#x5264;&#x306E;&#x52B9;&#x679C;&#x306B;&#x3088;&#x308B; tgf &beta; &#x306E;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x4E0A;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x6271;&#x308F; tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x57F9;&#x990A;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306E;&#x767A;&#x73FE;&#x3092;&#x89B3;&#x5BDF;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x52B9;&#x679C;&#x306F;&#x8981; Smad4 &#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;Tgfbeta2(-/-)/Tgfbeta3(-/-) &#x4E8C;&#x91CD;&#x907A;&#x4F1D;&#x5B50;&#x5909;&#x7570;&#x30DE;&#x30A6;&#x30B9;&#x3092;&#x7528;&#x3044;&#x305F; in vivo &#x640D;&#x5931;&#x95A2;&#x6570;&#x89E3;&#x6790;&#x9AD8;&#x3081;&#x3089;&#x308C;&#x305F;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x306E;&#x53CD;&#x5BFE;&#x306E;&#x52B9;&#x679C;&#x3068;&#x5C11;&#x306A;&#x3044;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x5927;&#x8133;&#x76AE;&#x8CEA;&#x304A;&#x3088;&#x3073;&#x6D77;&#x99AC;&#x3067;&#x793A;&#x3057;&#x305F;&#x3002;Gata2&#x3001;Runx1&#x3001;&#x304A;&#x3088;&#x3073; Nedd9 &#x306B;&#x3088;&#x308B; tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x898F;&#x5236;&#x3057;&#x3001;&#x795E;&#x7D4C;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x767A;&#x9054;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x95A2;&#x4E0E;&#x304C;&#x77E5;&#x3089;&#x308C;&#x308B;&#x5019;&#x88DC;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x3060;&#x3063;&#x305F;&#x3002;SiRNA &#x3092;&#x4ECB;&#x3057;&#x305F;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005; Nedd9 &#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x904B;&#x547D;&#x306E; tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x4F9D;&#x5B58;&#x6027;&#x306E;&#x5897;&#x52A0;&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3068;&#x3057;&#x3066;&#x8B58;&#x5225;&#x3002;Tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x6CBB;&#x7642;&#x306F;&#x3001;&#x3057;&#x304B;&#x3057;&#x307E;&#x305F; tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x9732;&#x51FA;&#x306E;&#x6642;&#x306B;&#x89B3;&#x6E2C;&#x3055;&#x308C;&#x305F; Nestin &#x967D;&#x6027;&#x524D;&#x99C6;&#x30D7;&#x30FC;&#x30EB;&#x306E;&#x5F62;&#x614B;&#x5909;&#x5316;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x5F8C; beta1 &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x7D4C;&#x8DEF;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x5206;&#x5B50;&#x3068;&#x3057;&#x3066;&#x4E3B;&#x306B;&#x8A18;&#x8F09;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x3053;&#x306E;&#x8DB3;&#x5834;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x306F;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;

&#x6210;&#x9577;&#x56E0;&#x5B50; &beta; &#x30DE;&#x30A6;&#x30B9;&#x76AE;&#x8CEA;&#x3068;&#x6D77;&#x99AC;&#x524D;&#x99C6;&#x7D30;&#x80DE; in vitro &#x304A;&#x3088;&#x3073; in vivo &#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;: &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3068;&#x3057;&#x3066;&#x306E; Nedd9 &#x306E;&#x540C;&#x5B9A;&#x3002;

&#xBCC0;&#xD615; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xBCA0;&#xD0C0; (Tgfbeta)&#xC640; &#xAD00;&#xB828; &#xB41C; &#xC2E0;&#xD638; &#xC774;&#xD399;&#xD130; &#xAC1C;, &#xD45C;&#xD604; &#xD558;&#xC9C0;&#xB9CC; &#xC57D;&#xAC04; &#xAC1C; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548;&#xC774; &#xB2E4;&#xAE30;&#xB2A5; cytokine&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xB300; &#xD55C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. &#xB9C8;&#xC6B0;&#xC2A4; &#xB450;&#xB1CC; &#xAC1C;&#xBC1C;&#xC758; hippocampal &#xBC0F; &#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC758; &#xC77C;&#xCC28; &#xC804;&#xC9C0; culture&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;&#xC774; &#xC5F0;&#xAD6C; Tgfbeta &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; &#xC720;&#xC804;&#xC790; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xC6B4;&#xBA85;&#xC758; &#xCC44;&#xD0DD; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; progenitors&#xC758; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xC885;&#xB8CC;&#xC640; &#xAD00;&#xB828; &#xB41C; &#xBC1C;&#xACAC;. &#xC774; &#xB530;&#xB77C; progenitors&#xC5D0; Tgfbeta&#xC758; antimitotic &#xC601;&#xD5A5; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; Tgfbeta &#xCE58;&#xB8CC; &#xBB38;&#xD654;&#xC5D0; &#xC2E0;&#xACBD; &#xB9C8;&#xCEE4;&#xC758; &#xC99D;&#xAC00; &#xC2DD;&#xC744; &#xAD00;&#xCC30;&#xD569;&#xB2C8;&#xB2E4;. &#xC774; &#xD6A8;&#xACFC; smad4&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, Tgfbeta2(-/-)/Tgfbeta3(-/-) &#xC774;&#xC911; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB9C8;&#xC6B0;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; in vivo &#xAE30;&#xB2A5; &#xC190;&#xC2E4; &#xBD84;&#xC11D; &#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xACFC; &#xD574; &#xB9C8;&#xC5D0; &#xC788;&#xB294; &#xC99D;&#xAC00; &#xB41C; &#xC138;&#xD3EC; &#xC99D;&#xC2DD;&#xC758; &#xBC18;&#xB300; &#xD6A8;&#xACFC; &#xC801;&#xC740; &#xB274;&#xB7F0;&#xC744; &#xBCF4;&#xC600;&#xB2E4;. Gata2, Runx1, Nedd9 &#xD6C4;&#xBCF4; &#xC720;&#xC804;&#xC790; Tgfbeta&#xC5D0; &#xC758;&#xD574; &#xD1B5;&#xC81C; &#xB418; &#xACE0; &#xC2E0;&#xACBD; progenitors&#xC758; &#xAC1C;&#xBC1C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xAC83;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xC5C8;&#xB2E4;. Nedd9 &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xC6B4;&#xBA85; Tgfbeta &#xC885;&#xC18D; &#xC99D;&#xAC00; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC778; &#xC2E0;&#xD638; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB85C; &#xC2DD;&#xBCC4; &#xCD5C;&#xC800;&#xC758; siRNA &#xC911;&#xC7AC;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC;. Tgfbeta &#xCE58;&#xB8CC; &#xD558;&#xC9C0;&#xB9CC; Tgfbeta&#xC5D0; &#xB178;&#xCD9C; &#xC2DC; &#xAD00;&#xCC30; Nestin &#xC591;&#xC131; &#xBFCC;&#xB9AC; &#xD480;&#xC758; &#xD615;&#xD0DC;&#xD559; &#xBCC0;&#xD654;&#xC5D0; &#xC5F0;&#xAD00;&#xB3C4; &#xD6C4; beta1 integrin &#xD1B5;&#xB85C;&#xC758; &#xC2E0;&#xD638; &#xBD84;&#xC790;&#xB3C4; &#xC8FC;&#xB85C; &#xC124;&#xBA85; &#xD558;&#xB294;,&#xC774; &#xC2A4; &#xCE90; &#xD3F4;&#xB529; &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD;&#xB9CC; &#xC720;&#xB3C4; &#xD558;&#xC9C0;.

&#xBCC0;&#xD615; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xBCA0;&#xD0C0; &#xCD09;&#xC9C4; &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xC6B4;&#xBA85;&#xC758; &#xB9C8;&#xC6B0;&#xC2A4;&#xC758; &#xB300;&#xB1CC; &#xD53C; &#xC9C8; &#xBC0F; hippocampal progenitors&#xC758; &#xC0DD;&#xCCB4; &#xC678; &#xBC0F; &#xC0DD;&#xCCB4; &#xC870;&#xAC74;: nedd9&#xC758; &#xD544;&#xC218;&#xC801;&#xC778; &#xC2E0;&#xD638; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB85C; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

Factor de crecimiento transformante beta (Tgfbeta) y efectores se&ntilde;alizaci&oacute;n asociadas se expresan en el cerebro anterior, pero poco se sabe sobre el papel de esta citoquina multifuncional durante el desarrollo del prosenc&eacute;falo. Mediante cultivos celulares de primaria hipocampal y cortical de desarrollo de cerebro de rat&oacute;n, este estudio identific&oacute; genes regulados por el Tgfbeta asociados no s&oacute;lo con la salida del ciclo celular de progenitores, sino con la adopci&oacute;n del destino celular neuronal. En consecuencia, observamos no s&oacute;lo un efecto antimit&oacute;tica de Tgfbeta en progenitores, sino tambi&eacute;n un incremento en la expresi&oacute;n de marcadores neuronales en culturas Tgfbeta tratado. Este efecto fue dependiente de Smad4. Adem&aacute;s, an&aacute;lisis de p&eacute;rdida de funci&oacute;n en vivo usando ratones mutantes dobles Tgfbeta2(-/-)/Tgfbeta3(-/-) demostraron el efecto opuesto de la proliferaci&oacute;n celular mayor y menos neuronas en la corteza cerebral y el hipocampo. Gata2, Runx1 y Nedd9 fueron los genes candidatos regulada por Tgfbeta y conocido por participar en los procesos de desarrollo de trasplante de precursores neuronales. Usando ca&iacute;da mediada por siRNA, identificamos Nedd9 como un componente esencial de la se&ntilde;alizaci&oacute;n para el aumento de Tgfbeta-dependiente en destino de c&eacute;lulas neuronales. Expresi&oacute;n de esta prote&iacute;na de andamiaje, que es principalmente descrita como una mol&eacute;cula de se&ntilde;alizaci&oacute;n de la v&iacute;a de la integrina beta1, no s&oacute;lo fue inducida despu&eacute;s de Tgfbeta tratamiento pero tambi&eacute;n se asoci&oacute; con cambios morfol&oacute;gicos de la piscina del progenitor Nestin positivo observado sobre la exposici&oacute;n a Tgfbeta.

Factor de crecimiento transformante beta promueve destino celular neuronal de progenitores con cortical e hipocampo de rat&oacute;n in vitro e in vivo: identificaci&oacute;n de Nedd9 como un componente esencial de la se&ntilde;alizaci&oacute;n.

Epigenetics: development, dynamics and disease.

Simultaneous generation of neural cells and that of the nutrient-supplying vasculature during brain development is called neurovascular coupling. We report on a transgenic mouse with impaired transforming growth factor β (TGFβ)-signalling in forebrain-derived neural cells using a Foxg1-cre knock-in to drive the conditional knock-out of the Tgfbr2. Although the expression of FOXG1 is assigned to neural progenitors and neurons of the telencephalon, Foxg1(cre/+);Tgfbr2(flox/flox) (Tgfbr2-cKO) mutants displayed intracerebral haemorrhage. Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching. Vascular endothelial growth factor (VEGF) A, insulin-like growth factor (IGF) 1, IGF2, TGFβ, inhibitor of DNA binding (ID) 1, thrombospondin (THBS) 2, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1 were altered in either expression levels or tissue distribution. Accordingly, human umbilical vein endothelial cells (HUVEC) displayed branching defects after stimulation with conditioned medium (CM) that was derived from primary neural cultures of the ventral and dorsal telencephalon of Tgfbr2-cKO. Supplementing CM of Tgfbr2-cKO with VEGFA rescued these defects, but application of TGFβ aggravated them. HUVEC showed reduced migration towards CM of mutants compared with controls. Supplementing the CM with growth factors VEGFA, fibroblast growth factor (FGF) 2 and IGF1 partially restored HUVEC migration. In contrast, TGFβ supplementation further impaired migration of HUVEC. We observed differences along the dorso-ventral axis of the telencephalon with regard to the impact of these factors on the phenotype. Together these data establish a TGFBR2-dependent molecular crosstalk between neural and endothelial cells during brain vessel development. These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.

Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.

Epigenetic regulatory mechanisms play a central role in controlling gene expression during development, cell differentiation and tumorigenesis. Monoubiquitination of histone H2B is one epigenetic modification which is dynamically regulated by the opposing activities of specific ubiquitin ligases and deubiquitinating enzymes (DUBs). The Ubiquitin-specific Protease 22 (USP22) is the ubiquitin hydrolase component of the human SAGA complex which deubiquitinates histone H2B during transcription. Recently, many studies have investigated an oncogenic potential of USP22 overexpression. However, its physiological function in organ maintenance, development and its cellular function remain largely unknown. A previous study reported embryonic lethality in Usp22 knockout mice. Here we describe a mouse model with a global reduction of USP22 levels which expresses the LacZ gene under the control of the endogenous Usp22 promoter. Using this reporter we found Usp22 to be ubiquitously expressed in murine embryos. Notably, adult Usp2(2lacZ/lacZ) displayed low residual Usp22 expression levels coupled with a reduced body size and weight. Interestingly, the reduction of Usp22 significantly influenced the frequency of differentiated cells in the small intestine and the brain while H2B and H2Bub1 levels remained constant. Taken together, we provide evidence for a physiological role for USP22 in controlling cell differentiation and lineage specification.

Usp22 deficiency impairs intestinal epithelial lineage specification in vivo.

Transforming growth factor β (TGFβ)-mediated anti-proliferative and differentiating effects promote neuronal differentiation during embryonic central nervous system development. TGFβ downstream signals, composed of activated SMAD2/3, SMAD4 and a FOXO family member, promote the expression of cyclin-dependent kinase inhibitor Cdkn1a. In early CNS development, IGF1/PI3K signaling and the transcription factor FOXG1 inhibit FOXO- and TGFβ-mediated Cdkn1a transcription. FOXG1 prevents cell cycle exit by binding to the SMAD/FOXO-protein complex. In this study we provide further details on the FOXG1/FOXO/SMAD transcription factor network. We identified ligands of the TGFβ- and IGF-family, Foxo1, Foxo3 and Kcnh3 as novel FOXG1-target genes during telencephalic development and showed that FOXG1 interferes with Foxo1 and Tgfβ transcription. Our data specify that FOXO1 activates Cdkn1a transcription. This process is under control of the IGF1-pathway, as Cdkn1a transcription increases when IGF1-signaling is pharmacologically inhibited. However, overexpression of CDKN1A and knockdown of Foxo1 and Foxo3 is not sufficient for neuronal differentiation, which is probably instructed by TGFβ-signaling. In mature neurons, FOXG1 activates transcription of the seizure-related Kcnh3, which might be a FOXG1-target gene involved in the FOXG1 syndrome pathology.

The FOXG1/FOXO/SMAD network balances proliferation and differentiation of cortical progenitors and activates Kcnh3 expression in mature neurons.

Dystonia musculorum is a neurodegenerative disorder caused by a mutation in the dystonin gene. It has been described in mice and humans where it is called hereditary sensory autonomic neuropathy. Mutated mice show severe movement disorders and die at the age of 3-4 weeks. This study describes the discovery and molecular, clinical, as well as pathological characterization of a new spontaneously occurring mutation in the dystonin gene in C57BL/6N mice. The mutation represents a 40-kb intragenic deletion allele of the dystonin gene on chromosome 1 with exactly defined deletion borders. It was demonstrated by Western blot, mass spectrometry, and immunohistology that mice with a homozygous mutation were entirely devoid of the dystonin protein. Pathomorphological lesions were restricted to the brain stem and spinal cord and consisted of swollen, argyrophilic axons and dilated myelin sheaths in the white matter and, less frequently, total chromatolysis of neurons in the gray matter. Axonal damage was detected by amyloid precursor protein and nonphosphorylated neurofilament immunohistology. Axonopathy in the central nervous system (CNS) represents the hallmark of this disease. Mice with the dystonin mutation also showed suppurative inflammation in the respiratory tract, presumably due to brain stem lesion-associated food aspiration, whereas skeletal muscles showed no pathomorphological changes. This study describes a novel mutation in the dystonin gene in mice leading to axonopathy in the CNS. In further studies, this model may provide new insights into the pathogenesis of neurodegenerative diseases and may elucidate the complex interactions of dystonin with various other cellular proteins especially in the CNS.

Axonopathy in the Central Nervous System Is the Hallmark of Mice with a Novel Intragenic Null Mutation of Dystonin.

The brain is made up of trillions of synaptic connections that together form neural networks needed for normal brain function and behavior. SLM2 is a member of a conserved family of RNA binding proteins, including Sam68 and SLM1, that control splicing of Neurexin1-3 pre-mRNAs. Whether SLM2 affects neural network activity is unknown. Here, we find that SLM2 levels are maintained by a homeostatic feedback control pathway that predates the divergence of SLM2 and Sam68. SLM2 also controls the splicing of Tomosyn2, LysoPLD/ATX, Dgkb, Kif21a, and Cask, each of which are important for synapse function. Cortical neural network activity dependent on synaptic connections between SLM2-expressing-pyramidal neurons and interneurons is decreased in Slm2-null mice. Additionally, these mice are anxious and have a decreased ability to recognize novel objects. Our data reveal a pathway of SLM2 homeostatic auto-regulation controlling brain network activity and behavior.

A SLM2 Feedback Pathway Controls Cortical Network Activity and Mouse Behavior.

Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.

Neuronal Activity, TGFβ-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus.

Tanja Vogel

Institute for Anatomy and Cell Biology,
Department of Molecular Embryology,
Faculty of Medicine,
Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine

University of Freiburg

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Tanja Vogel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Anatomy and Cell Biology,Department of Molecular Embryology,Faculty of Medicine,Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine

University of Freiburg

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Institute for Anatomy and Cell Biology,
Department of Molecular Embryology,
Faculty of Medicine,
Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine