generation-aggregates-mouse-embryonic-stem-cells-that-show-symmetry

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour <em>In Vitro</em>

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

University of Cambridge

Dorsal closure is a morphogenetic process involving the coordinated convergence of two epithelial sheets to enclose the Drosophila melanogaster embryo. Specialized populations of cells at the edges of each epithelial sheet, the dorsal-most epidermal cells, emit actin-based processes that are essential for the proper enclosure of the embryo. Here we show that actin dynamics at the leading edge is preceded by a planar polarization of the dorsal-most epidermal cells associated with a reorganization of the cytoskeleton. An important consequence of this planar polarization is the formation of actin-nucleating centres at the leading edge, which are important in the dynamics of actin. We show that Wingless (Wg) signalling and Jun amino-terminal kinase (JNK) signalling have overlapping but different roles in these events.

Planar polarity and actin dynamics in the epidermis of Drosophila.

&#x5EFA;&#x7ACB;&#x548C;&#x7EC6;&#x80DE;&#x547D;&#x8FD0;&#x5728;&#x53D1;&#x5C55;&#x671F;&#x95F4;&#x7684;&#x7A33;&#x5B9A;&#x53D6;&#x51B3;&#x4E8E;&#x591A;&#x4E2A;&#x4FE1;&#x53F7;&#xFF0C;&#x6700;&#x7EC8;&#x8C03;&#x8282;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x5177;&#x4F53;&#x6A21;&#x5F0F;&#x7684;&#x6574;&#x5408;&#x3002;&#x867D;&#x7136;&#x6709;&#x5145;&#x5206;&#x7684;&#x8BC1;&#x636E;&#x8868;&#x660E;&#x6B64;&#x96C6;&#x6210;&#x4E00;&#x7EA7;&#x7684;&#x57FA;&#x56E0;&#x8C03;&#x63A7;&#x5E8F;&#x5217;&#xFF0C;&#x4E86;&#x89E3;&#x751A;&#x5C11;&#x5176;&#x4ED6;&#x5404;&#x7EA7;&#x7684;&#x7EC6;&#x80DE;&#x6D3B;&#x6027;&#x53CA;&#x5176;&#x64CD;&#x4F5C;&#x3002;Wnt &#x548C;&#x69FD;&#x53E3;&#x4FE1;&#x53F7;&#x662F;&#x8C03;&#x8282;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x53D1;&#x5C55;&#x548C;&#x75BE;&#x75C5;&#x7684;&#x7535;&#x8DEF;&#x7684;&#x91CD;&#x8981;&#x56E0;&#x7D20;&#x3002;&#x9057;&#x4F20;&#x5206;&#x6790;&#x8868;&#x660E;&#x9664;&#x4E86;&#x5BF9;&#x7279;&#x5B9A;&#x57FA;&#x56E0;&#x7684;&#x8F6C;&#x5F55;&#x6536;&#x655B;&#xFF0C;&#x6709;&#x8C03;&#x8282;&#x7684;&#x4EA4;&#x53C9;&#x76D1;&#x7BA1;&#x4E92;&#x52A8;&#x8FD9;&#x4E9B;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x901A;&#x8DEF;&#x3002;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x8FD9;&#x4E9B;&#x4EA4;&#x4E92;&#x8282;&#x70B9;&#x7684;&#x4F4D;&#x7F6E;&#x662F;&#x6D3B;&#x52A8;&#x7684;&#x6D3B;&#x52A8;&#x548C;&#x6D3B;&#x52A8;/&#x7624;&#x72B0;&#x72F3;&#x8FDE;&#x73AF;&#x7684;&#x7A97;&#x4F53;&#x7684;&#x6570;&#x91CF;&#x4F5C;&#x4E86;&#x89C4;&#x5B9A;&#x7684;&#x7F3A;&#x53E3;&#x3002;&#x51F9;&#x69FD;&#x7684;&#x8FD9;&#x9879;&#x6D3B;&#x52A8;&#x662F;&#x72EC;&#x7ACB;&#x7684;&#x8BF1;&#x5BFC;&#x5176;&#x80DE;&#x5185;&#x57DF;&#x7684;&#x5288;&#x88C2;&#x548C;&#x5176;&#x4E2D;&#x4ECB;&#x5BFC;&#x8F6C;&#x5F55;&#x901A;&#x8FC7;&#x82CF; (H) / CBF1&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6240;&#x8FF0;&#x7684;&#x69FD;&#x53E3;&#x7684;&#x8C03;&#x8282;&#x529F;&#x80FD;&#x6709;&#x52A9;&#x4E8E; Wnt &#x4FE1;&#x53F7;&#x53EF;&#x80FD;&#x626E;&#x6F14;&#x91CD;&#x8981;&#x7684;&#x89D2;&#x8272;&#xFF0C;&#x5728;&#x6B63;&#x5E38;&#x548C;&#x75C5;&#x7406;&#x60C5;&#x51B5;&#x4E0B;&#x5EFA;&#x7ACB;&#x7684;&#x9C81;&#x68D2;&#x7684;&#x9608;&#x503C;&#x3002;

&#x51F9;&#x69FD;&#x8C03;&#x8282; Wnt &#x4FE1;&#x53F7;&#x7531;&#x5173;&#x8054;&#x72B0;&#x72F3;&#x8FDE;&#x73AF;&#x5E76;&#x8C03;&#x8282;&#x5176;&#x8F6C;&#x5F55;&#x6D3B;&#x6027;&#x3002;

The establishment and stability of cell fates during development depend on the integration of multiple signals, which ultimately modulate specific patterns of gene expression. While there is ample evidence for this integration at the level of gene regulatory sequences, little is known about its operation at other levels of cellular activity. Wnt and Notch signalling are important elements of the circuitry that regulates gene expression in development and disease. Genetic analysis has suggested that in addition to convergence on the transcription of specific genes, there are modulatory cross-regulatory interactions between these signalling pathways. We report that the nodal point of these interactions is an activity of Notch that regulates the activity and the amount of the active/oncogenic form of Armadillo/beta-catenin. This activity of Notch is independent of that induced upon cleavage of its intracellular domain and which mediates transcription through Su(H)/CBF1. The modulatory function of Notch described here, contributes to the establishment of a robust threshold for Wnt signalling which is likely to play important roles in both normal and pathological situations.

Notch modulates Wnt signalling by associating with Armadillo/beta-catenin and regulating its transcriptional activity.

La mise en place et la stabilit&eacute; des destins cellulaires au cours du d&eacute;veloppement d&eacute;pendent de l&#39;int&eacute;gration des signaux multiples, qui modulent en fin de compte des mod&egrave;les pr&eacute;cis de l&#39;expression g&eacute;nique. Bien qu&#39;il y a suffisamment de preuves pour cette int&eacute;gration au niveau des s&eacute;quences r&eacute;gulatrices du g&egrave;ne, on en sait peu sur ses activit&eacute;s &agrave; d&#39;autres niveaux de l&#39;activit&eacute; cellulaire. Wnt et la signalisation Notch sont des &eacute;l&eacute;ments importants de la circuiterie qui r&eacute;gule l&#39;expression des g&egrave;nes dans le d&eacute;veloppement et la maladie. L&#39;analyse g&eacute;n&eacute;tique a sugg&eacute;r&eacute; qu&#39;en plus de convergence sur la transcription de g&egrave;nes sp&eacute;cifiques, il y a des interactions modulatrices Croix r&eacute;glementaires entre ces voies de signalisation. Les auteurs rapportent que le point nodal de ces interactions est une activit&eacute; de la prot&eacute;ine Notch qui r&eacute;glemente l&#39;activit&eacute; et la quantit&eacute; de forme active/oncog&egrave;nes de tatou/b&ecirc;ta-cat&eacute;nine. Cette activit&eacute; de la prot&eacute;ine Notch est ind&eacute;pendante de celle induite par clivage de son domaine intracellulaire et qui intervient dans la transcription par l&#39;interm&eacute;diaire Su (H) / CBF1. La fonction modulatrice de la prot&eacute;ine Notch d&eacute;crite ici, contribue &agrave; l&#39;&eacute;tablissement d&#39;un seuil robuste pour Wnt de signalisation qui sont susceptibles de jouer un r&ocirc;le important dans les situations normales et pathologiques.

Encoche module de signalisation Wnt en associant avec tatou/b&ecirc;ta-cat&eacute;nine et en r&eacute;glementant son activit&eacute; transcriptionnelle.

Notch moduliert Wnt-Signalweg durch den Umgang mit Armadillo / beta-Catenin und Regulierung ihrer transkriptionellen Aktivit&#xE4;t.

L&#39;istituzione e la stabilit&agrave; dei destini delle cellule durante lo sviluppo dipendono l&#39;integrazione di diversi segnali, che in definitiva modulare specifici pattern di espressione genica. Mentre ci sono ampie prove per questa integrazione a livello di sequenze di geni regolatori, poco si sa circa il funzionamento ad altri livelli di attivit&agrave; cellulare. WNT e segnalazione Notch sono elementi importanti del circuito che regola l&#39;espressione genica in sviluppo e malattia. L&#39;analisi genetica ha suggerito che oltre alla convergenza sulla trascrizione di geni specifici, esistono modulatrice interazioni cross-normative tra queste vie di segnalazione. Segnaliamo che il punto nodale di queste interazioni &egrave; un&#39;attivit&agrave; di Notch che regola l&#39;attivit&agrave; e la quantit&agrave; della forma attiva/oncogenica di Armadillo/beta-catenina. Questa attivit&agrave; di Notch &egrave; indipendente da quella indotta sulla scissione del suo dominio intracellulare e che da intermediario trascrizione attraverso Su (H) / CBF1. La funzione modulatrice della tacca qui descritto, contribuisce alla creazione di una robusta soglia per Wnt segnalazione che &egrave; probabile che giocano un ruolo importante in situazioni normali e patologiche.

Tacca modula Wnt segnalazione da associare Armadillo/beta-catenina e regolamentare l&#39;attivit&agrave; trascrizionale.

&#x6700;&#x7D42;&#x7684;&#x306B;&#x7279;&#x5B9A;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x8907;&#x6570;&#x306E;&#x4FE1;&#x53F7;&#x306E;&#x7D71;&#x5408;&#x306F;&#x3001;&#x8A2D;&#x7ACB;&#x3068;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x958B;&#x767A;&#x6642;&#x306E;&#x5B89;&#x5B9A;&#x6027;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x5236;&#x5FA1;&#x914D;&#x5217;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x7D71;&#x5408;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5341;&#x5206;&#x306A;&#x8A3C;&#x62E0;&#x304C;&#x3042;&#x308B;&#x304C;&#x3001;&#x307B;&#x3068;&#x3093;&#x3069;&#x305D;&#x306E;&#x4ED6;&#x7D30;&#x80DE;&#x306E;&#x6D3B;&#x52D5;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x64CD;&#x4F5C;&#x306B;&#x3064;&#x3044;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;Wnt &#x3068;&#x30CE;&#x30C3;&#x30C1; &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x958B;&#x767A;&#x3068;&#x75BE;&#x60A3;&#x306B;&#x304A;&#x3051;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x56DE;&#x8DEF;&#x306E;&#x91CD;&#x8981;&#x306A;&#x8981;&#x7D20;&#x3067;&#x3059;&#x3002;&#x907A;&#x4F1D;&#x5B66;&#x7684;&#x89E3;&#x6790;&#x306F;&#x3001;&#x7279;&#x5B9A;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8EE2;&#x5199;&#x306E;&#x53CE;&#x675F;&#x306B;&#x52A0;&#x3048;&#x3066;&#x306F;&#x3001;&#x306A;&#x97F3;&#x306E;&#x30AF;&#x30ED;&#x30B9;&#x898F;&#x5236;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3053;&#x308C;&#x3089;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x7D50;&#x7BC0;&#x70B9;&#x306E;&#x6D3B;&#x52D5;&#x3068;&#x30A2;&#x30EB;&#x30DE;&#x30B8;&#x30ED;/&beta;-&#x30AB;&#x30C6;&#x30CB;&#x30F3;&#x306E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;/&#x767A;&#x764C;&#x5F62;&#x306E;&#x91CF;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x30CE;&#x30C3;&#x30C1;&#x306E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30CE;&#x30C3;&#x30C1;&#x306E;&#x3053;&#x306E;&#x6D3B;&#x52D5;&#x306F;&#x3001;&#x305D;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x5375;&#x5272;&#x6642;&#x306B;&#x8A98;&#x8D77;&#x3055;&#x308C;&#x308B;&#x306E;&#x72EC;&#x7ACB;&#x3057;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x8EE2;&#x5199; Su (H) &#x3092;&#x901A;&#x3058;&#x3066;&#x3092;&#x4EF2;&#x4ECB;/CBF1&#x3002;&#x3053;&#x3053;&#x3067;&#x8AAC;&#x660E;&#x306E;&#x30CE;&#x30C3;&#x30C1;&#x306E;&#x97F3;&#x6A5F;&#x80FD;&#x306F;&#x6B63;&#x5E38;&#x304A;&#x3088;&#x3073;&#x75C5;&#x7684;&#x4E21;&#x65B9;&#x306E;&#x72B6;&#x6CC1;&#x3067;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x518D;&#x751F;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308B; wnt &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x5805;&#x7262;&#x306A;&#x3057;&#x304D;&#x3044;&#x5024;&#x306E;&#x78BA;&#x7ACB;&#x306B;&#x8CA2;&#x732E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30CE;&#x30C3;&#x30C1; &#x30A2;&#x30EB;&#x30DE;&#x30B8;&#x30ED;/&beta;-&#x30AB;&#x30C6;&#x30CB;&#x30F3;&#x3068;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3001;&#x305D;&#x306E;&#x8EE2;&#x5199;&#x6D3B;&#x6027;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B; Wnt &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3092;&#x5909;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC124;&#xB9BD; &#xBC0F; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xC140; &#xC6B4;&#xBA85; &#xC548;&#xC815;&#xC131; &#xAD81;&#xADF9;&#xC801;&#xC73C;&#xB85C; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC758; &#xD2B9;&#xC815; &#xD328;&#xD134;&#xC744; &#xC870;&#xC808; &#xD558;&#xB294; &#xC5EC;&#xB7EC; &#xC2E0;&#xD638; &#xD1B5;&#xD569;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xADDC;&#xC815; &#xC2DC;&#xD000;&#xC2A4;&#xC758; &#xC218;&#xC900;&#xC5D0;&#xC11C;&#xC774; &#xD1B5;&#xD569;&#xC5D0; &#xB300; &#xD55C; &#xCDA9;&#xBD84; &#xD55C; &#xC99D;&#xAC70;&#xAC00; &#xC788;&#xC9C0;&#xB9CC;, &#xC791;&#xC740; &#xC138;&#xD3EC; &#xD65C;&#xB3D9;&#xC758; &#xB2E4;&#xB978; &#xC218;&#xC900;&#xC5D0;&#xC11C;&#xC758; &#xC791;&#xC5C5;&#xC5D0; &#xB300; &#xD55C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. Wnt &#xBC0F; &#xB178;&#xCE58; &#xC2E0;&#xD638; &#xC804;&#xB2EC; &#xAC1C;&#xBC1C; &#xBC0F; &#xC9C8;&#xBCD1;&#xC758; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC744; &#xC870;&#xC808; &#xD558;&#xB294; &#xD68C;&#xB85C;&#xC758; &#xC911;&#xC694; &#xD55C; &#xC694;&#xC18C;&#xC785;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xBD84;&#xC11D;&#xC740; &#xD2B9;&#xC815; &#xC720;&#xC804;&#xC790;&#xC758; &#xC804;&#xC0AC;&#xC5D0; &#xCEE8;&#xBC84;&#xC804;&#xC2A4; &#xC774;&#xC678;&#xC5D0; &#xC774;&#xB7EC;&#xD55C; &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xC0AC;&#xC774; modulatory &#xD06C;&#xB85C;&#xC2A4; &#xADDC;&#xC81C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC81C;&#xC548; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xAC00; &#xC774;&#xB7EC;&#xD55C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xAFB8;&#xBC85;&#xAFB8;&#xBC85; &#xC878; &#xAE30; &#xD3EC;&#xC778;&#xD2B8; &#xD65C;&#xC131;/&#xAE08;&#xD558;&#xB294; &#xD615;&#xD0DC;&#xC758; &#xC544; &#xB974; &#xB9C8; &#xB51C;/&#xBCA0;&#xD0C0;-catenin&#xC758; &#xC591;&#xACFC; &#xD65C;&#xB3D9;&#xC744; &#xC870;&#xC808; &#xD558;&#xB294; &#xB178;&#xCE58;&#xC758; &#xD65C;&#xB3D9; &#xBCF4;&#xACE0; &#xD569;&#xB2C8;&#xB2E4;. &#xB178;&#xCE58;&#xC758;&#xC774; &#xD65C;&#xB3D9;&#xC758; &#xC138;&#xD3EC;&#xB0B4; &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xC720; &#xBC29; &#xC0AC;&#xC774;&#xC758; &#xACE8;&#xC9DC;&#xAE30;&#xB97C; &#xB530;&#xB77C; &#xC720;&#xB3C4; &#xD558;&#xB294; &#xB3C5;&#xB9BD;&#xC801;&#xC774; &#xACE0;&#xB294; &#xC804;&#xC0AC; &#xC4F0; (H)&#xC744; &#xD1B5;&#xD574; &#xC911;&#xC7AC; / CBF1. &#xC5EC;&#xAE30;&#xC11C; &#xC124;&#xBA85; &#xD558;&#xB294; &#xB178;&#xCE58;&#xC758; modulatory &#xD568;&#xC218; Wnt &#xC2E0;&#xD638;&#xB294; &#xC815;&#xC0C1;&#xC801;&#xC774; &#xACE0; &#xBCD1; &#xC801;&#xC778; &#xC0C1;&#xD669;&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;&#xC5D0; &#xB300; &#xD55C; &#xAC15;&#xB825;&#xD55C; &#xC784;&#xACC4;&#xAC12;&#xC758; &#xC124;&#xB9BD;&#xC5D0; &#xAE30;&#xC5EC;.

&#xB178;&#xCE58; &#xC544; &#xB974; &#xB9C8; &#xB51C;/&#xBCA0;&#xD0C0;-catenin&#xC5D0; &#xC5F0;&#xACB0; &#xD558; &#xACE0; &#xADF8;&#xAC83;&#xC758; transcriptional &#xD65C;&#xB3D9;&#xC744; &#xADDC;&#xC81C; &#xD558; &#xC5EC; Wnt &#xC2E0;&#xD638; &#xBCC0;&#xC870;.

El establecimiento y la estabilidad de los sinos de c&eacute;lulas durante el desarrollo dependen de la integraci&oacute;n de se&ntilde;ales m&uacute;ltiples, que en &uacute;ltima instancia modulan patrones espec&iacute;ficos de la expresi&oacute;n g&eacute;nica. Aunque existe amplia evidencia para esta integraci&oacute;n a nivel de secuencias reguladoras de genes, poco se conoce sobre su funcionamiento en otros niveles de la actividad celular. Wnt y muesca de se&ntilde;alizaci&oacute;n son elementos importantes de los circuitos que regula la expresi&oacute;n g&eacute;nica en el desarrollo y la enfermedad. El an&aacute;lisis gen&eacute;tico ha sugerido que adem&aacute;s de convergencia en la transcripci&oacute;n de genes espec&iacute;ficos, hay interacciones regulatorias cruzadas moduladores entre estas v&iacute;as de se&ntilde;alizaci&oacute;n. Se reporta que el punto nodal de estas interacciones es una actividad de la muesca que regula la actividad y la cantidad de la forma activa/oncog&eacute;nico de Armadillo/beta-catenina. Esta actividad de la muesca es independiente de que indujo al escote de su dominio intracelular y que media la transcripci&oacute;n a trav&eacute;s de Su (H) / CBF1. La funci&oacute;n de modulador de la muesca descrito aqu&iacute;, contribuye al establecimiento de un umbral robusto para Wnt se&ntilde;alizaci&oacute;n que es probable que desempe&ntilde;an papeles importantes en situaciones normales y patol&oacute;gicas.

Muesca modula la se&ntilde;alizaci&oacute;n de Wnt asociar con Armadillo/beta-catenina y regulando su actividad transcripcional.

The Notch gene of Drosophila encodes a single transmembrane receptor that plays a central role in the process of lateral inhibition. This process results in the selection of individual mesodermal and neural precursors during the development of the muscular and nervous systems. The activation of Notch during lateral inhibition is mediated by the transmembrane ligand Delta (Dl) and effected by the transcription factor Suppressor of Hairless (Su(H)). The same functional cassette plays a role in other processes, in particular, the development and patterning of the wing. Genetic analysis has suggested that, in addition to the Su(H)-dependent pathway, Notch can signal in an Su(H)-independent manner. This process seems to be tightly associated with signalling by Wingless, a member of the Wnt family of signalling molecules. Here, we have analyzed further the possibility that the Notch protein encodes two different functions. To do so, we have studied the activities and genetic properties of different Notch receptors bearing deletions of specific regions of the intracellular and the extracellular domains in different developmental processes, and have sought to correlate the activity of these mutant proteins with those of existing mutants in Notch. Our results support the existence of at least two different activities of Notch each of which can be associated with specific structural domains.

Notch receptor encodes two structurally separable functions in Drosophila: a genetic analysis.

Cell fate decisions require the integration of various signalling inputs at the level of transcription and signal transduction. Wnt and Notch signalling are two important signalling systems that operate in concert in a variety of systems in vertebrates and invertebrates. There is evidence that the Notch receptor can modulate Wnt signalling and that its target is the activity and levels of Armadillo/beta-catenin. Here, we characterize this function of Notch in relation to Axin, a key element in the regulation of Wnt signalling that acts as a scaffold for the Shaggy/GSK3beta-dependent phosphorylation of Armadillo/beta-catenin. While Notch can regulate ectopic Wingless signalling caused by loss of function of Shaggy, it can only partially regulate the ectopic Wnt signalling induced by the loss of Axin function. The same interactions are observed in tissue culture cells where we observe a synergy in between Axin and Notch in the regulation of Armadillo/beta-catenin. Our results provide evidence for a function of Axin in the regulation of Armadillo that is different from its role as a scaffold for GSK3beta.

Notch synergizes with axin to regulate the activity of armadillo in Drosophila.

Notch receptors act as ligand-dependent membrane-tethered transcription factors with a prominent role in binary cell fate decisions during development, which is conserved across species. In addition there is increasing evidence for other functions of Notch, particularly in connection with Wnt signalling: Notch is able to modulate the activity of Armadillo/ss-catenin, the effector of Wnt signalling, in a manner that is independent of its transcriptional activity. Here we explore the mechanism of this interaction in the epithelium of the Drosophila imaginal discs and find that it is mediated by the ligand-independent endocytosis and traffic of the Notch receptor. Our results show that Notch associates with Armadillo near the adherens junctions and that it is rapidly endocytosed promoting the traffic of an activated form of Armadillo into endosomal compartments, where it may be degraded. As Notch has the ability to interact with and downregulate activated forms of Armadillo, it is possible that in vivo Notch regulates the transcriptionally competent pool of Armadillo. These interactions reveal a previously unknown activity of Notch, which serves to buffer the function of activated Armadillo and might underlie some of its transcription-independent effects.

Ligand-independent traffic of Notch buffers activated Armadillo in Drosophila.

Here we investigate the structural and functional basis of the interactions between Notch and Wingless signalling in Drosophila. Using yeast-two-hybrid and pull-down assays we show that Notch can bind directly a form of Dishevelled that is stabilized upon Wingless signalling. Moreover, we show that the mechanism by which Wingless signalling is able to downregulate Notch is by promoting its ligand-independent traffic to a compartment where it is degraded and that this activity depends on Dishevelled.

Wingless modulates the ligand independent traffic of Notch through Dishevelled.

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of β-catenin. Here, we show that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module.

Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of 'self-renewal' and 'differentiation' genes. Here, we combine single-cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post-translational regulation in the maintenance of pluripotency, which complements the well-established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and β-catenin suffices to account for the behavior of ES cells under different conditions. Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.

A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells.

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'.

Tina Balayo

Department of Genetics

University of Cambridge

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro