induction-and-analysis-of-epithelial-to-mesenchymal-transition

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

R&D Systems, Inc.

&#x5728;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x7684;&#x65E9;&#x671F;&#x975E;&#x6D32;&#x722A;&#x87FE;&#x80CC;&#x8F74;&#x7531; Wnt &#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x901A;&#x8DEF;&#xFF0C;&#x6D89;&#x53CA;&#x8FDE;&#x73AF;&#x8FD0;&#x5165;&#x80CC;&#x7EC6;&#x80DE;&#x6838;&#x548C;&#x5176;&#x4E0E;&#x5DE6;&#x952E;&#x578B;&#x8F6C;&#x5F55;&#x56E0;&#x5B50; XTcf3 &#x7684;&#x529F;&#x80FD;&#x5173;&#x8054;&#x6307;&#x5B9A;&#x3002;&#x968F;&#x540E;&#x7684; XTcf3 &#x7684;&#x51FD;&#x6570;&#x662F;&#x4E0D;&#x786E;&#x5B9A;&#x7684;&#x3002;&#x8FC7;&#x5EA6;&#x8868;&#x8FBE;&#x6570;&#x636E;&#x8868;&#x660E;&#x5B83;&#x53EF;&#x4EE5;&#x6D3B;&#x5316;&#x5242;&#x548C;&#x963B;&#x904F;&#x7269;&#x7684;&#x4E0B;&#x6E38;&#x57FA;&#x56E0;&#x3002;XTcf3 mRNA &#x5408;&#x6210;&#x5728;&#x975E;&#x6D32;&#x722A;&#x87FE;&#x5375;&#x5B50;&#x53D1;&#x751F;&#x8FC7;&#x7A0B;&#x548C;&#x5B58;&#x50A8;&#x5728;&#x86CB;&#x4E2D;&#x3002;&#x82E5;&#x8981;&#x8BC6;&#x522B;&#x5176;&#x80CC;&#x8F74;&#x89C4;&#x8303;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x6211;&#x4EEC;&#x8017;&#x5C3D;&#x6B64;&#x4EA7;&#x5987;&#x5B58;&#x50A8;&#x5728;&#x4F7F;&#x7528;&#x53CD;&#x4E49; deoxyoligonucleotides&#xFF0C;&#x6210;&#x719F;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x548C;&#x5B83;&#x4EEC;&#x53D7;&#x7CBE;&#x3002;XTcf3 &#x8017;&#x5C3D;&#xFF0C;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x548C;&#x6807;&#x8BB0;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x4E0A;&#x7684;&#x53D1;&#x5C55;&#x7684;&#x5F71;&#x54CD;&#x4E3B;&#x8981;&#x662F;&#x663E;&#x793A;&#xFF0C;XTcf3 &#x662F;&#x4E00;&#x79CD;&#x6291;&#x5236;&#x5242;&#xFF0C;&#x9632;&#x6B62;&#x540E;&#x671F; blastula &#x5355;&#x5143;&#x683C;&#x80CC;&#x4FA7;&#x548C;&#x8179;&#x4FA7;&#x80CC;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x663E;&#x793A;&#x7B80;&#x5355;&#x6551;&#x6D4E;&#x7684;&#x9547;&#x538B;&#x4E0D;&#x662F;&#x6240;&#x9700;&#x7684;&#x80CC;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x552F;&#x4E00;&#x56E0;&#x7D20;&#x3002;&#x4E3A;&#x4E86;&#x8868;&#x660E;&#x8FD9;&#x4E00;&#x70B9;&#xFF0C;&#x6211;&#x4EEC;&#x53D7;&#x7CBE;&#x5375;&#xFF0C;&#x5DF2;&#x88AB;&#x7834;&#x574F;&#x6B86;&#x5C3D;&#x7684; XTcf3 &#x548C; VegT &#x7684;&#x4EA7;&#x5987;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x3002;&#x901A;&#x5E38;&#x7531; XTcf3 &#x538B;&#x6291;&#x7684;&#x80CC;&#x57FA;&#x56E0;&#x4E0D;&#x4F1A;&#x6FC0;&#x6D3B;&#x8FD9;&#x4E9B;&#x80DA;&#x80CE;&#x4E2D;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E;&#x6B63;&#x5E38;&#x80CC;&#x57FA;&#x56E0;&#x5728;&#x80DA;&#x80CE;&#x7684;&#x8868;&#x8FBE;&#x9700;&#x8981;&#x8F6C;&#x5F55;&#x6D3B;&#x5316;&#x5242; VegT&#xFF0C;&#x867D;&#x7136; XTcf3 &#x53EF;&#x4EE5;&#x9632;&#x6B62;&#x4ED6;&#x4EEC;&#x4E0D;&#x6070;&#x5F53;&#x8868;&#x8FBE;&#x8179;&#x4E00;&#x4FA7;&#x7684;&#x80DA;&#x80CE;&#x3002;

&#x80CC;&#x4FA7;&#x548C;&#x8179;&#x4FA7;&#x975E;&#x6D32;&#x722A;&#x87FE;&#x7EC6;&#x80DE;&#x4ECB;&#x5BFC;&#x4EA7;&#x5987; XTcf3 &#x7EC4;&#x7EC7;&#x8005;&#x57FA;&#x56E0;&#x7684;&#x9547;&#x538B;&#x3002;

In the early Xenopus embryo, the dorsal axis is specified by a Wnt signal transduction pathway, involving the movement of beta-catenin into dorsal cell nuclei and its functional association with the LEF-type transcription factor XTcf3. The subsequent function of XTcf3 is uncertain. Overexpression data has suggested that it can be both an activator and repressor of downstream genes. XTcf3 mRNA is synthesized during oogenesis in Xenopus and is stored in the egg. To identify its role in dorsal axis specification, we depleted this maternal store in full-grown oocytes using antisense deoxyoligonucleotides, and fertilized them. The developmental effects of XTcf3 depletion, both on morphogenesis and the expression of marker genes, show that primarily, XTcf3 is an inhibitor, preventing both dorsal and ventral cells of the late blastula from expressing dorsal genes. We also show that simple relief from the repression is not the only factor required for dorsal gene expression. To demonstrate this, we fertilized eggs that had been depleted of both XTcf3 and the maternal transcription factor VegT. Dorsal genes normally repressed by XTcf3 are not activated in these embryos. These data show that normal dorsal gene expression in the embryo requires the transcriptional activator VegT, whilst XTcf3 prevents their inappropriate expression on the ventral side of the embryo.

Repression of organizer genes in dorsal and ventral Xenopus cells mediated by maternal XTcf3.

Dans l&#x26;#39;embryon de Xenopus, l&#x26;#39;axe dorsale est sp&#xE9;cifi&#xE9;e par une voie de transduction de signal Wnt, impliquant le d&#xE9;placement de la beta-cat&#xE9;nine dans le noyau des cellules dorsale et son association fonctionnelle avec le facteur de transcription LEF-type de XTcf3. La fonction ult&#xE9;rieure XTcf3 est incertain. La surexpression de donn&#xE9;es a sugg&#xE9;r&#xE9; qu&#x26;#39;il puisse &#xEA;tre &#xE0; la fois un activateur et r&#xE9;presseur des g&#xE8;nes en aval. XTcf3 ARNm est synth&#xE9;tis&#xE9; au cours de l&#x26;#39;ovogen&#xE8;se chez le X&#xE9;nope et est stock&#xE9;e dans l&#x26;#39;&#x153;uf. Pour identifier son r&#xF4;le dans la sp&#xE9;cification axe dorsal, nous appauvri ce magasin maternelle &#xE0; plein cultiv&#xE9;es en utilisant des ovocytes d&#xE9;soxyoligonucl&#xE9;otides antisens, et les f&#xE9;cond&#xE9;. Les effets sur le d&#xE9;veloppement de l&#x26;#39;&#xE9;puisement XTcf3, &#xE0; la fois sur la morphogen&#xE8;se et l&#x26;#39;expression des g&#xE8;nes marqueurs, principalement montrent que, XTcf3 est un inhibiteur, la pr&#xE9;vention de deux cellules dorsales et ventrales de la fin des ann&#xE9;es blastula de l&#x26;#39;expression de g&#xE8;nes dorsales. Nous montrons &#xE9;galement que l&#x26;#39;all&#xE9;gement de simple &#xE0; partir de la r&#xE9;pression n&#x26;#39;est pas le seul facteur n&#xE9;cessaire pour l&#x26;#39;expression des g&#xE8;nes dorsale. Pour d&#xE9;montrer cela, nous avons des &#x153;ufs f&#xE9;cond&#xE9;s qui avaient &#xE9;t&#xE9; &#xE9;puis&#xE9;s &#xE0; la fois XTcf3 et la transcription Vegt maternelle facteur. G&#xE8;nes dorsaux normalement r&#xE9;prim&#xE9;s par XTcf3 ne sont pas activ&#xE9;s dans ces embryons. Ces donn&#xE9;es montrent que l&#x26;#39;expression des g&#xE8;nes normale dorsale de l&#x26;#39;embryon exige l&#x26;#39;activateur transcriptionnel Vegt, tandis que XTcf3 emp&#xEA;che leur expression inappropri&#xE9;e sur la face ventrale de l&#x26;#39;embryon.

R&#xE9;pression des g&#xE8;nes dans les cellules de Xenopus Organisateur dorsale et ventrale M&#xE9;diation par XTcf3 maternelle

Die Repression von Genen Veranstalter in dorsalen und ventralen Xenopus-Zellen vermittelt durch m&#xFC;tterliche XTcf3.

Nell&#39;embrione di Xenopus precoce, l&#39;asse dorsale &egrave; specificato da un percorso trasduzione del segnale Wnt, che coinvolge il movimento di beta-catenina in nuclei delle cellule dorsale e la sua associazione funzionale con il fattore di trascrizione LEF-tipo XTcf3. La funzione successiva del XTcf3 &egrave; incerta. Dati di sovraespressione ha suggerito che pu&ograve; essere sia un attivatore e repressore dei geni a valle. XTcf3 mRNA &egrave; sintetizzato durante l&#39;ovogenesi in Xenopus e viene memorizzato nell&#39;uovo. Per identificare il proprio ruolo nella specifica di asse dorsale, impoverito questo negozio materno nell&#39;adulto ovociti mediante antisenso deoxyoligonucleotides e fecondato li. Gli effetti dello sviluppo della deplezione di XTcf3, sia sulla morfogenesi e l&#39;espressione dei geni marcatori, mostrano che, soprattutto, XTcf3 &egrave; un inibitore, impedendo l&#39;espressione dei geni dorsale dorsale e ventrale le cellule della blastula fine. Mostriamo anche che semplice sollievo dalla repressione non &egrave; l&#39;unico fattore richiesto per l&#39;espressione di gene dorsale. Per dimostrare questo, abbiamo fecondato le uova che erano state impoverite sia XTcf3 che il fattore di trascrizione materna VegT. Dorsale geni normalmente represse dal XTcf3 non vengono attivati in questi embrioni. Questi dati mostrano che espressione di gene normale dorsale nell&#39;embrione richiede l&#39;attivatore trascrizionale VegT, mentre XTcf3 impedisce la loro espressione inappropriata sul lato ventrale dell&#39;embrione.

Repressione di Organizzatore geni nelle cellule di Xenopus dorsale e ventrale mediate da XTcf3 materna.

&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x521D;&#x671F;&#x80DA;&#x3067;&#x306F;&#x3001;&#x80CC;&#x5074;&#x8EF8;&#x306F;&#x80CC;&#x5074;&#x7D30;&#x80DE;&#x6838;&#x3068;LEF&#x578B;&#x8EE2;&#x5199;&#x56E0;&#x5B50;XTcf3&#x3068;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x95A2;&#x9023;&#x306B;&#x3B2;-&#x30AB;&#x30C6;&#x30CB;&#x30F3;&#x306E;&#x79FB;&#x52D5;&#x3092;&#x4F34;&#x3046;Wnt&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306B;&#x3088;&#x3063;&#x3066;&#x6307;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; XTcf3&#x306E;&#x5F8C;&#x7D9A;&#x306E;&#x6A5F;&#x80FD;&#x306F;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308B;&#x3002;&#x904E;&#x5270;&#x767A;&#x73FE;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x4E0B;&#x6D41;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x6291;&#x5236;&#x306E;&#x4E21;&#x65B9;&#x304C;&#x53EF;&#x80FD;&#x306A;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002; XTcf3 mRNA&#x3092;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x306E;&#x5375;&#x5F62;&#x6210;&#x306E;&#x9593;&#x306B;&#x5408;&#x6210;&#x3055;&#x308C;&#x3066;&#x304A;&#x308A;&#x3001;&#x5375;&#x306B;&#x683C;&#x7D0D;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x80CC;&#x8EF8;&#x4ED5;&#x69D8;&#x3067;&#x305D;&#x306E;&#x5F79;&#x5272;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x30A2;&#x30F3;&#x30C1;&#x30BB;&#x30F3;&#x30B9;&#x30C7;&#x30AA;&#x30AD;&#x30B7;&#x30AA;&#x30EA;&#x30B4;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x5B8C;&#x5168;&#x306B;&#x6210;&#x9577;&#x3057;&#x305F;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x3001;&#x3053;&#x306E;&#x6BCD;&#x89AA;&#x306E;&#x5E97;&#x3092;&#x67AF;&#x6E07;&#x3057;&#x3001;&#x305D;&#x308C;&#x3089;&#x3092;&#x53D7;&#x7CBE;&#x3002;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x3068;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306E;&#x4E21;&#x65B9;XTcf3&#x67AF;&#x6E07;&#x306E;&#x767A;&#x9054;&#x3078;&#x306E;&#x5F71;&#x97FF;&#x306F;&#x3001;&#x4E3B;&#x306B;&#x3001;XTcf3&#x306F;&#x80CC;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x767A;&#x73FE;&#x3059;&#x308B;&#x304B;&#x3089;&#x9045;&#x304F;&#x80DE;&#x80DA;&#x306E;&#x80CC;&#x5074;&#x3068;&#x8179;&#x5074;&#x7D30;&#x80DE;&#x306E;&#x4E21;&#x65B9;&#x3092;&#x9632;&#x6B62;&#x3059;&#x308B;&#x305F;&#x3081;&#x3001;&#x963B;&#x5BB3;&#x5264;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3002;&#x8868;&#x793A;&#x307E;&#x305F;&#x3001;&#x6291;&#x5727;&#x304B;&#x3089;&#x306E;&#x5358;&#x7D14;&#x306A;&#x6551;&#x6E08;&#x304C;&#x80CC;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x552F;&#x4E00;&#x306E;&#x8981;&#x56E0;&#x3067;&#x306F;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x3053;&#x308C;&#x3092;&#x5B9F;&#x8A3C;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;XTcf3&#x3068;&#x6BCD;&#x6027;&#x8EE2;&#x5199;&#x56E0;&#x5B50;VegT&#x4E21;&#x65B9;&#x304C;&#x67AF;&#x6E07;&#x3057;&#x3066;&#x3044;&#x305F;&#x5375;&#x3092;&#x53D7;&#x7CBE;&#x3002;&#x901A;&#x5E38;XTcf3&#x306B;&#x3088;&#x3063;&#x3066;&#x6291;&#x5236;&#x80CC;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x80DA;&#x3067;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x80DA;&#x306E;&#x6B63;&#x5E38;&#x306A;&#x80CC;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306F;&#x3001;&#x8EE2;&#x5199;&#x6D3B;&#x6027;&#x5316;&#x56E0;&#x5B50;VegT&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3001;&#x306A;&#x304C;&#x3089;XTcf3&#x306F;&#x3001;&#x80DA;&#x306E;&#x8179;&#x5074;&#x306B;&#x5F7C;&#x3089;&#x306E;&#x4E0D;&#x9069;&#x5207;&#x306A;&#x767A;&#x73FE;&#x3092;&#x9632;&#x3050;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

&#x6BCD;&#x4F53;XTcf3&#x306B;&#x3088;&#x3063;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x308B;&#x80CC;&#x8179;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x306E;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AA;&#x30FC;&#x30AC;&#x30CA;&#x30A4;&#x30B6;&#x30FC;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5F3E;&#x5727;

&#xCD08;&#xAE30; Xenopus &#xBC30;&#xC544; &#xC9C0; &#xCD95; Wnt &#xC2E0;&#xD638; &#xC804;&#xB2EC; &#xD1B5;&#xB85C;, &#xC9C0; &#xC138;&#xD3EC; &#xD575;&#xC758; &#xAE30;&#xB2A5; &#xC5F0;&#xAD00; LEF &#xD615; &#xC804;&#xC0AC; &#xC778;&#xC790; Xtcf3&#xC73C;&#xB85C; &#xBCA0;&#xD0C0;-catenin&#xC758; &#xC6C0;&#xC9C1;&#xC784;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC9C0;&#xC815; &#xB429;&#xB2C8;&#xB2E4;. Xtcf3&#xC758; &#xD6C4;&#xC18D; &#xAE30;&#xB2A5; &#xBD88;&#xD22C;&#xBA85; &#xD558;&#xB2E4;&#xC785;&#xB2C8;&#xB2E4;. Overexpression &#xB370;&#xC774;&#xD130;&#xB294; &#xD65C;&#xC131; &#xC81C; &#xBC0F; &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xC720;&#xC804;&#xC790;&#xC758; &#xC9C4;&#xC555; &#xB420; &#xC218; &#xC81C;&#xC548; &#xD588;&#xB2E4;. XTcf3 mRNA Xenopus&#xC5D0;&#xC11C; oogenesis &#xC911; &#xD569;&#xC131; &#xD558; &#xACE0; &#xACC4;&#xB780;&#xC5D0; &#xC800;&#xC7A5; &#xB429;&#xB2C8;&#xB2E4;. &#xC9C0; &#xCD95; &#xC0AC;&#xC591; &#xD558;&#xB294; &#xC5ED;&#xD560;&#xC744; &#xC2DD;&#xBCC4; &#xD558; &#xACE0;&#xAC08; &#xC13C;&#xC2A4; deoxyoligonucleotides&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC804;&#xCCB4; oocytes&#xC5D0;&#xC11C; &#xBAA8;&#xC131;&#xC774; &#xAC8C; &#xD558; &#xACE0; &#xADF8;&#xB4E4;&#xC744; &#xC218;&#xC815; &#xB41C;. XTcf3 &#xACE0;&#xAC08;, &#xBAA8;&#xB450; morphogenesis &#xB9C8;&#xCEE4; &#xC720;&#xC804;&#xC790;&#xC758; &#xD45C;&#xD604;&#xC5D0;&#xC758; &#xBC1C;&#xB2EC; &#xD6A8;&#xACFC; &#xC8FC;&#xB85C;, XTcf3 &#xB4F1; &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604;&#xC5D0;&#xC11C; &#xB2A6;&#xC740; blastula&#xC758; &#xB4F1; &#xCABD;&#xACFC; &#xBCF5; &#xBD80; &#xC140;&#xC744; &#xBC29;&#xC9C0; &#xD55C; &#xC5B5;&#xC81C;&#xC81C;&#xB294; &#xBCF4;&#xC5EC;&#xC900;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xC5B5;&#xC555;&#xC5D0;&#xC11C; &#xAC04;&#xB2E8;&#xD55C; &#xAD6C;&#xD638; &#xB4F1; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xC720;&#xC77C;&#xD55C; &#xC694;&#xC18C;&#xAC00; &#xC544;&#xB2C8;&#xB2E4;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC774; &#xC124;&#xBA85; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC; Xtcf3&#xACFC; &#xBAA8;&#xC131; &#xC804;&#xC0AC; &#xC778;&#xC790; VegT &#xACE0;&#xAC08; &#xB418;&#xC5B4; &#xD588;&#xB2E4; &#xACC4;&#xB780; &#xC218;&#xC815; &#xB41C;. &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; Xtcf3&#xC5D0; &#xC758;&#xD574; &#xC5B5;&#xC555; &#xD558;&#xB294; &#xB4F1; &#xC720;&#xC804;&#xC790; &#xC774;&#xB7EC;&#xD55C; &#xBC30;&#xC544;&#xC5D0;&#xC11C; &#xD65C;&#xC131;&#xD654; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xD45C;&#xC2DC; XTcf3 &#xD0DC;&#xC544;&#xC758; &#xBCF5; &#xBD80; &#xCABD;&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xBD80;&#xC801;&#xC808; &#xD55C; &#xD45C;&#xD604;&#xC744; &#xBC29;&#xC9C0; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD0DC;&#xC544;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC9C0; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604; transcriptional &#xD65C;&#xC131;&#xAE30; VegT, &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;.

&#xB4F1; &#xCABD;&#xACFC; &#xBCF5; &#xBD80; Xenopus &#xC140; &#xBAA8;&#xC131; Xtcf3&#xC5D0; &#xC758;&#xD574; &#xC911;&#xC7AC;&#xC5D0; &#xC8FC;&#xCD5C;&#xC790; &#xC720;&#xC804;&#xC790;&#xC758; &#xC5B5;&#xC81C;.

A principios de los embriones de Xenopus, el eje dorsal se especifica por una v&#xED;a de transducci&#xF3;n de se&#xF1;ales Wnt, que implica el movimiento de la beta-catenina en los n&#xFA;cleos celulares dorsal y su relaci&#xF3;n funcional con el factor de transcripci&#xF3;n LEF tipo XTcf3. La funci&#xF3;n posterior de XTcf3 es incierta. Datos sobreexpresi&#xF3;n ha sugerido que puede ser tanto un activador y represor de genes abajo. XTcf3 ARNm se sintetiza durante la ovog&#xE9;nesis en Xenopus y se almacena en el huevo. Para identificar su papel en la especificaci&#xF3;n del eje dorsal, que agota esta tienda materna en plena crecido ovocitos utilizando desoxioligonucle&#xF3;tidos antisentido, y se fertilizan ellos. Los efectos sobre el desarrollo de XTcf3 agotamiento, tanto en la morfog&#xE9;nesis y la expresi&#xF3;n de los genes marcadores, muestran que en primer lugar, XTcf3 es un inhibidor, la prevenci&#xF3;n de las dos c&#xE9;lulas dorsal y ventral de la bl&#xE1;stula tard&#xED;a de la expresi&#xF3;n de genes dorsales. Tambi&#xE9;n muestran que el alivio de simple a partir de la represi&#xF3;n no es el &#xFA;nico factor necesario para la expresi&#xF3;n del gen dorsal. Para demostrar esto, los huevos fertilizados que se hab&#xED;a agotado de tanto XTcf3 y el factor de transcripci&#xF3;n Vegt materna. Genes dorsales normalmente reprimidos por XTcf3 no se activan en los embriones. Estos datos muestran que la expresi&#xF3;n de gen normal dorsal en el embri&#xF3;n requiere el activador transcripcional vEGT, mientras XTcf3 impide su expresi&#xF3;n inapropiada en el lado ventral del embri&#xF3;n.

La represi&#xF3;n de los genes en las c&#xE9;lulas del organizador de Xenopus dorsal y ventral mediada por la madre XTcf3

K+ channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (K Ca ) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+ channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung K Ca channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, K Ca channel expression is increased and TASK is decreased.

Pulmonary vascular K+ channel expression and vasoreactivity in a model of congenital heart disease.

&#x8981;&#x6D4B;&#x8BD5;&#x8BE5;&#x6162;&#x6027;&#x5BAB;&#x5185;&#x80BA;&#x52A8;&#x8109;&#x9AD8;&#x538B; (PHTN) &#x59A5;&#x534F; (PA) &#x80BA;&#x52A8;&#x8109;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE; (SMC) O2 &#x9065;&#x611F;&#x3001; &#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x7528;&#x6765;&#x7814;&#x7A76; Po2 &#x6025;&#x6027;&#x589E;&#x52A0;&#x5BF9;&#x80DE;&#x6D46; Ca2 + &#x6D53;&#x5EA6;&#x7684;&#x5F71;&#x54CD;&#x7684;&#x5047;&#x8BBE; ([Ca2 +] &#x6211;) PA SMC &#x5728;&#x539F;&#x4EE3;&#x57F9;&#x517B;&#x957F;&#x671F;&#x7F3A;&#x6C27; subconfluent &#x5355;&#x5206;&#x5B50;&#x819C;&#x3002;PA &#x6821;&#x8463;&#x4F1A;&#x53D6;&#x81EA;&#x80CE;&#x7F8A;&#x4E0E; PHTN &#x7531;&#x4E8E;&#x5BAB;&#x5185;&#x7ED3;&#x624E;&#x7684;&#x52A8;&#x8109;&#x5BFC;&#x7BA1;&#x672A;&#x95ED;&#x3002;&#x6025;&#x6027;&#x5E38;&#x51CF;&#x5C11; [Ca2 +] i &#x7684;&#x63A7;&#x4EF6;&#xFF0C;&#x4F46;&#x4E0D;&#x662F; PHTN PA SMC&#x3002;&#x5728;&#x63A7;&#x4EF6;&#x4E2D; PA SMC&#x3001; [Ca2 +] &#x6211;&#x4EE5;&#x540E; Ca2 + &#x589E;&#x52A0;-&#x654F;&#x611F; (&#x8BE5;&#x9879;&#x76EE;) &#x548C;&#x7535;&#x538B;&#x654F;&#x611F; (Kv) K + &#x901A;&#x9053;&#x5C01;&#x9501;&#x548C;&#x5730;&#x5C14;&#x786B;&#x6CBB;&#x7597;&#x540E;&#x4E0B;&#x964D;&#x3002;&#x5728; PHTN PA SMC&#x3001; &#x8BE5;&#x9879;&#x76EE;&#x5C01;&#x9501;&#x6CA1;&#x6709;&#x4EFB;&#x4F55;&#x6548;&#x679C;&#xFF0C;&#x800C; Kv &#x5C01;&#x9501;&#x4E0E;&#x786B;&#x6C2E;&#x589E;&#x52A0; [Ca2 +] &#x4E00;&#x3001; &#x6291;&#x5236;&#x808C;&#x6D46;&#x7F51; Ca2 + ATPase &#x6D3B;&#x6027;&#x5BFC;&#x81F4; [Ca2 +] &#x4E2D;&#x7684;&#x589E;&#x52A0;&#x4E86;&#x66F4;&#x591A;&#x7684;&#x6211;&#x5728;&#x63A7;&#x4EF6;&#x4E2D;&#x4E0E;&#x6BD4;&#x8F83; PHTN PA SMC&#x3002;&#x76F8;&#x53CD;&#xFF0C;&#x9020;&#x6210;&#x66F4;&#x5927;&#x7684;&#x5170;&#x5C3C;&#x78B1;&#x589E;&#x52A0; [Ca2 +] &#x6211;&#x5728; PHTN &#x4E0E;&#x63A7;&#x5236;&#x5DF4;&#x52D2;&#x65AF;&#x5766;&#x6743;&#x529B;&#x673A;&#x6784; SMC &#x76F8;&#x6BD4;&#x3002;&#x8BE5;&#x9879;&#x76EE;&#x901A;&#x9053; mRNA &#x51CF;&#x5C11;&#x548C; mRNA &#x4E0D;&#x53D8;&#x7684;&#x5728; PHTN PA SMC &#x7684; Kv &#x901A;&#x9053;&#x4E0E;&#x63A7;&#x4EF6;&#x76F8;&#x6BD4;&#x3002;&#x6211;&#x4EEC;&#x5F97;&#x51FA;&#x7684;&#x7ED3;&#x8BBA; PHTN &#x59A5;&#x534F; PA SMC O2 &#x9065;&#x611F;&#x3001; &#x6539;&#x53D8;&#x7EC6;&#x80DE;&#x5185; Ca2 + &#x7A33;&#x6001;&#xFF0C;&#x548C;&#x66F4;&#x6539;&#x7684;&#x4E3B;&#x8981;&#x79BB;&#x5B50;&#x901A;&#x9053;&#x7684;&#x786E;&#x5B9A;&#x57FA;&#x5E95; [Ca2 +] &#x6211;&#x4ECE; Kv &#x5230;&#x8BE5;&#x9879;&#x76EE;&#x3002;

&#x6162;&#x6027;&#x5BAB;&#x5185;&#x80BA;&#x52A8;&#x8109;&#x9AD8;&#x538B;&#x6709;&#x635F;&#x80CE;&#x513F;&#x7684;&#x80BA;&#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE; O2 &#x7684;&#x4F20;&#x611F;&#x3002;

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.

Chronic intrauterine pulmonary hypertension compromises fetal pulmonary artery smooth muscle cell O2 sensing.

Pour v&eacute;rifier l&#39;hypoth&egrave;se que cette chronique hypertension pulmonaire intra-ut&eacute;rine (PHTN) compromet l&#39;art&egrave;re pulmonaire (AP) muscle lisse cellulaire (SMC) O2 sentant, microscopie de fluorescence a &eacute;t&eacute; utilis&eacute;e pour &eacute;tudier l&#39;effet d&#39;une augmentation aigu&euml; Po2 sur la concentration de Ca2 + cytosolique ([Ca2 +] i) de hypoxiques monocouches sont des PA SMC en culture primaire. PA SMCs provenaient des agneaux foetaux avec PHTN en raison de la ligature du canal art&eacute;riel intra-ut&eacute;rine. Normoxie aigu&euml; a diminu&eacute; [Ca2 +] i en contr&ocirc;le, mais pas PHTN PA SMC. Contr&ocirc;le PA SMC, [Ca2 +] j&#39;ai augment&eacute; apr&egrave;s la Ca2 +-d&eacute;pendantes (KCa) et sensibles &agrave; la tension (Kv) K + channel blocus et a diminu&eacute; apr&egrave;s traitement de diltiazem. Dans PHTN PA SMC, KCa blocus n&#39;a eu aucun effet, alors que diltiazem et Kv blocus ont augment&eacute; [Ca2 +] i. Inhibition du r&eacute;ticulum sarcoplasmique Ca2 + ATPasique a provoqu&eacute; une augmentation de plus de [Ca2 +] i dans les contr&ocirc;les par rapport &agrave; PHTN PA SMC. &Agrave; l&#39;inverse, ryanodine caus&eacute;e une plus grande augmentation de [Ca2 +] i PHTN comparativement aux t&eacute;moins PA SMC. Canaux KCa Qu&#39;arnm est diminu&eacute;e et Kv Qu&#39;arnm est inchang&eacute;e dans PHTN PA SMC comparativement aux t&eacute;moins. Nous concluons que PHTN compromet la d&eacute;tection PA SMC O2, alt&egrave;re l&#39;hom&eacute;ostasie du Ca2 + intracellulaire et change le canal ionique pr&eacute;dominante qui d&eacute;termine basale [Ca2 +] i de KCa &agrave; Kv.

L&#39;hypertension pulmonaire chronique intra-ut&eacute;rine compromet le muscle lisse de l&#39;art&egrave;re pulmonaire f&oelig;tale cellule O2 d&eacute;tection.

Testen Sie die Hypothese, dass chronische intrauterine Lungenhochdruck (PHTN) Kompromisse Pulmonalarterie (PA) Glattmuskel Zelle (SMC) O2 Sensorik, Fluoreszenzmikroskopie wurde verwendet, um den Effekt von einem akuten Anstieg der Po2 auf der cytosolic Ca2 +-Konzentration ([Ca2 +] ich) der chronisch hypoxic subconfluent Monolayers PA SMC in der Prim&auml;rkultur. PA SMCs stammen aus fetalen L&auml;mmer mit PHTN durch intrauterine Verbindung der Ductus arteriosus Botalli. Akute Normoxia zur&uuml;ckgegangen [Ca2 +] i-Steuerelement jedoch nicht PHTN PA SMC. Im Steuerelement PA SMC, [Ca2 +] i nach Ca2 erh&ouml;ht +-Sensitive (KCa) und Spannung-empfindlichen (Kv) K + Kanal-Blockade und sank nach Diltiazem Behandlung. PHTN PA SMC KCa-Blockade hatte keine Wirkung, Kv-Blockade und Diltiazem [Ca2 +] angehoben i. Hemmung der sarkoplasmatischen Retikulum Ca2 +-ATPase-Aktivit&auml;t verursacht einen gr&ouml;&szlig;eren Anstieg [Ca2 +] ich in Steuerelementen gegen&uuml;ber PHTN PA SMC. Umgekehrt Ryanodin verursacht eine gr&ouml;&szlig;ere Zunahme der [Ca2 +] i PHTN verglichen mit PA-SMC-Steuerelement. KCa-Kanal, die mRNA verringert wird und Kv-Kanal, die mRNA in PHTN PA SMC unver&auml;ndert ist verglichen mit Steuerelementen. Wir schlie&szlig;en, dass PHTN PA SMC O2 Sensorik Kompromisse, &auml;ndert der intrazellul&auml;ren Ca2 +-Hom&ouml;ostase und &auml;ndert die vorherrschende Ionenkanal, der bestimmt, basale [Ca2 +] i vom KCa zu Kv.

Chronischer intrauterine pulmonaler Hypertonie gef&auml;hrdet fetalen Lungenarterie glatten Muskelzellen Zelle O2 Sensorik.

Per testare l&#39;ipotesi che cronica ipertensione polmonare intrauterino (PHTN) compromette l&#39;arteria polmonare (PA) muscolo liscio cellula (SMC) O2 sensing, microscopia di fluorescenza &egrave; stata usata per studiare l&#39;effetto di un aumento acuto della Po2 sulla concentrazione di Ca2 + citosolico ([Ca2 +] i) di monostrati subconfluent cronicamente ipossiche di PA SMC in coltura primaria. PA SMCs derivavano da agnelli fetali con PHTN dovuto intrauterina legatura del dotto arterioso. Fenotipi acuta &egrave; diminuito [Ca2 +] i nel controllo, ma non PHTN PA SMC. Nel controllo PA SMC, [Ca2 +] ho aumentato dopo Ca2 +-sensitive (KCa) e sensibile tensione (Kv) K + canale blocco e diminuito dopo il trattamento di diltiazem. In PHTN PA SMC, blocco KCa ha avuto alcun effetto, mentre diltiazem e Kv blocco aumentato [Ca2 +] i. inibizione del reticolo sarcoplasmico attivit&agrave; Ca2 + ATPasi ha causato un maggiore aumento di [Ca2 +] i controlli rispetto ai PHTN PA SMC. Al contrario, rianodinico ha causato un maggiore aumento di [Ca2 +] I PHTN rispetto al controllo PA SMC. KCa che mRNA &egrave; diminuito e Kv canale che mRNA &egrave; immutato in PHTN PA SMC rispetto ai controlli. Concludiamo PHTN compromette PA SMC O2 sensing, altera l&#39;omeostasi del Ca2 + intracellulare che cambia il canale ionico predominante che determina basale [Ca2 +] i da KCa per Kv.

Ipertensione polmonare cronica intrauterina compromette arteria polmonare fetale smooth muscle cell O2 sensing.

&#x305D;&#x306E;&#x6162;&#x6027;&#x5B50;&#x5BAE;&#x5185;&#x80CE;&#x5150;&#x306E;&#x80BA;&#x9AD8;&#x8840;&#x5727; (PHTN) &#x3092;&#x59A5;&#x5354;&#x80BA;&#x52D5;&#x8108; (PA) &#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE; (SMC) O2 &#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x3001;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1; Po2 &#x306E;&#x6025;&#x6027;&#x5897;&#x52A0;&#x7D30;&#x80DE;&#x5185; ca 2 + &#x6FC3;&#x5EA6;&#x306E;&#x52B9;&#x679C;&#x7814;&#x7A76;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x305F;&#x4EEE;&#x8AAC;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3059;&#x308B;&#x306B;&#x306F; ([ca 2 +] I) PA SMC &#x306E;&#x6162;&#x6027;&#x7684;&#x4F4E;&#x9178;&#x7D20;&#x306E; subconfluent &#x819C;&#x521D;&#x4EE3;&#x57F9;&#x990A;&#x306E;&#x3002;PA SMCs PHTN &#x5B50;&#x5BAE;&#x52D5;&#x8108;&#x7BA1;&#x958B;&#x5B58;&#x7D50;&#x7D2E;&#x306B;&#x3088;&#x308B;&#x80CE;&#x5B50;&#x7F8A;&#x304B;&#x3089;&#x6D3E;&#x751F;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6025;&#x6027;&#x6642;&#x306E;&#x8133;&#x5185;&#x3092;&#x6E1B;&#x5C11; [ca 2 +] &#x306E;&#x5236;&#x5FA1;&#x304C;&#x306A;&#x3044; PHTN PA SMC &#x79C1;&#x306F;&#x3002;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB; PA SMC &#x79C1;&#x306E;&#x5897;&#x52A0;&#x5F8C;&#x306B; ca 2 + [ca 2 +]-&#x654F;&#x611F;&#x306A; (KCa) &#x3068;&#x96FB;&#x5727;&#x306B;&#x654F;&#x611F;&#x306A; (Kv) K + &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x5C01;&#x9396;&#x3068;&#x30B8;&#x30EB;&#x30C1;&#x30A2;&#x30BC;&#x30E0;&#x6CBB;&#x7642;&#x5F8C;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;PHTN PA SMC KCa &#x5C01;&#x9396;&#x306B;&#x52B9;&#x679C;&#x304C;&#x306A;&#x304B;&#x3063;&#x305F;&#x3001;Kv &#x5C01;&#x9396;&#x3068;&#x30B8;&#x30EB;&#x30C1;&#x30A2;&#x30BC;&#x30E0; [ca 2 +] &#x5897;&#x52A0;&#x3057;&#x305F;&#x306B;&#x5BFE;&#x3057; i. &#x7B4B;&#x5C0F;&#x80DE;&#x4F53; [ca 2 +] &#x3067;&#x5927;&#x304D;&#x304F;&#x5897;&#x52A0;&#x306B;&#x3088;&#x308B; ca 2 + atp &#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x963B;&#x5BB3;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x3067;&#x79C1;&#x306F; PHTN PA SMC &#x3068;&#x6BD4;&#x8F03;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x9006;&#x306B;&#x3001;&#x5927;&#x304D;&#x3044;&#x539F;&#x56E0;&#x30EA;&#x30A2;&#x30CE;&#x30B8;&#x30F3;&#x5897;&#x52A0; [ca 2 +] &#x306E;&#x79C1;&#x306F;&#x5236;&#x5FA1; PA SMC &#x6BD4;&#x8F03; PHTN &#x3067;&#x3002;MRNA &#x3092;&#x6E1B;&#x5C11; KCa &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x3068; mRNA PHTN PA SMC &#x3067;&#x5909;&#x66F4;&#x3055;&#x308C;&#x305A; Kv &#x30C1;&#x30E3;&#x30CD;&#x30EB; &#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x3068;&#x306E;&#x6BD4;&#x8F03;&#x3002;&#x6211;&#x3005; PHTN &#x59A5;&#x5354; PA SMC O2 &#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x3001;&#x7D30;&#x80DE;&#x5185; ca 2 + &#x6052;&#x5E38;&#x6027;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x3068;&#x3001;&#x57FA;&#x5E95; [ca 2 +] &#x6C7A;&#x5B9A;&#x512A;&#x52E2;&#x306A;&#x30A4;&#x30AA;&#x30F3; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x304C;&#x5909;&#x66F4;&#x3092;&#x7DE0;&#x7D50;&#x79C1;&#x306F; KCa Kv &#x306B;&#x304B;&#x3089;&#x3002;

&#x6162;&#x6027;&#x5B50;&#x5BAE;&#x5185;&#x80CE;&#x5150;&#x80BA;&#x9AD8;&#x8840;&#x5727;&#x75C7;&#x80CE;&#x5150;&#x306E;&#x80BA;&#x52D5;&#x8108;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE; O2 &#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x3092;&#x59A5;&#x5354;&#x3057;&#x307E;&#x3059;&#x3002;

&#xADF8; &#xB9CC;&#xC131; &#xC790;&#xAD81;&#xB0B4; &#xD3D0; &#xB3D9;&#xB9E5; &#xACE0;&#xD608;&#xC555; (PHTN) &#xD3D0; &#xB3D9;&#xB9E5; (PA) &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xC138;&#xD3EC; (SMC) o 2 &#xAC10;&#xC9C0;, &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95; cytosolic Ca2 + &#xB18D;&#xB3C4;&#xC5D0; Po2 &#xAE09;&#xC131; &#xC99D;&#xAC00;&#xC758; &#xD6A8;&#xACFC; &#xC5F0;&#xAD6C; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9;&#xD55C; &#xD0C0;&#xD611; &#xD558;&#xB294; &#xAC00;&#xC124;&#xC744; &#xD14C;&#xC2A4;&#xD2B8; ([Ca2 +] &#xB098;) &#xAE30;&#xBCF8; &#xBB38;&#xD654;&#xC5D0;&#xC11C; PA SMC&#xC758; &#xB9CC;&#xC131; hypoxic subconfluent monolayers&#xC758;. PA SMCs ductus arteriosus&#xC758; &#xC790;&#xAD81;&#xB0B4; &#xACB0; &#xCC30; &#xC778; PHTN&#xC640; &#xD568;&#xAED8; &#xD0DC;&#xC544; &#xC591;&#xC5D0;&#xC11C; &#xD30C;&#xC0DD; &#xB418;&#xC5C8;&#xB2E4;. &#xAE09;&#xC131; normoxia &#xAC10;&#xC18C; [Ca2 +] &#xC81C;&#xC5B4; &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; PHTN PA SMC&#xC5D0; &#xB098;. &#xCEE8;&#xD2B8;&#xB864;&#xC5D0;&#xC11C; PA SMC [Ca2 +] &#xD6C4; Ca2 + &#xC99D;&#xAC00;-&#xAD6C;&#xBD84; (KCa) &#xBC0F; &#xC804;&#xC555; (Kv) +&#xC5D0; &#xBBFC;&#xAC10;&#xD55C; &#xCC44;&#xB110; &#xBD09;&#xC1C4; &#xBC0F; diltiazem &#xCE58;&#xB8CC; &#xD6C4; &#xAC10;&#xC18C;. PHTN PA SMC KCa &#xBD09;&#xC1C4; &#xD6A8;&#xACFC;&#xAC00; &#xC5C6; &#xC5C8;, Kv &#xBD09;&#xC1C4; diltiazem &#xC99D;&#xAC00; [Ca2 +] &#xBC18;&#xBA74; i. sarcoplasmic &#xADF8;&#xBB3C; Ca2 + ATPase &#xD65C;&#xB3D9; [Ca2 +] &#xD070; &#xC99D;&#xAC00;&#xAC00; &#xBC1C;&#xC0DD; &#xC5B5;&#xC81C; &#xB098; &#xCEE8;&#xD2B8;&#xB864;&#xC5D0; PHTN PA SMC&#xC640; &#xBE44;&#xAD50;. &#xBC18;&#xB300;&#xB85C; ryanodine &#xD070; &#xBC1C;&#xC0DD; &#xC99D;&#xAC00;&#xC758; [Ca2 +] &#xB098; PHTN &#xC81C;&#xC5B4; PA SMC&#xC640; &#xBE44;&#xAD50;. KCa &#xCC44;&#xB110; mRNA &#xAC10;&#xC18C; &#xD558; &#xACE0; k v &#xCC44;&#xB110; PHTN PA SMC&#xC5D0;&#xC11C; Mrna&#xB97C; &#xBCC0;&#xACBD; &#xD558;&#xC9C0; &#xC54A;&#xACE0; &#xCEE8;&#xD2B8;&#xB864;&#xC5D0; &#xBE44;&#xD574;. &#xC6B0;&#xB9AC; &#xACB0;&#xB860; PHTN PA SMC o 2 &#xAC10;&#xC9C0; &#xD0C0;&#xD611; &#xC138;&#xD3EC;&#xB0B4; Ca2 + &#xD56D;&#xC0C1;&#xC131;, &#xBCC0;&#xACBD; &#xBC0F; &#xBCC0;&#xACBD; &#xACB0;&#xC815; &#xAE30;&#xC800; [Ca2 +] &#xC8FC;&#xB41C; &#xC774;&#xC628; &#xCC44;&#xB110; k V&#xC5D0;&#xB294; Kca&#xC5D0;&#xC11C; &#xB098;.

&#xD3D0; &#xB3D9;&#xB9E5; &#xACE0;&#xD608;&#xC555; &#xB9CC;&#xC131; &#xC790;&#xAD81;&#xB0B4; &#xD0DC;&#xC544; &#xD3D0; &#xB3D9;&#xB9E5; &#xD3C9;&#xD65C; &#xADFC; &#xC138;&#xD3EC; o 2 &#xAC10;&#xC9C0; &#xD0C0;&#xD611;.

Para probar la hip&oacute;tesis que hipertensi&oacute;n pulmonar cr&oacute;nica intrauterina (PHTN) compromete la arteria pulmonar (AP) del m&uacute;sculo liso (SMC) de la c&eacute;lula O2 sensor, microscop&iacute;a de fluorescencia se utiliz&oacute; para estudiar el efecto de un aumento agudo en la Po2 en la concentraci&oacute;n del Ca2 + citos&oacute;lico ([Ca2 +] i) de monocapas subconfluente hip&oacute;xicas cr&oacute;nico de SMC de PA en cultivo primario. PA SMCs se derivan de corderos fetales con PHTN debido a la ligadura intrauterina del ductus arterioso. Normoxia aguda disminuci&oacute;n [Ca2 +] i en control pero no PHTN PA SMC. Control SMC PA, [Ca2 +] aument&oacute; despu&eacute;s de Ca2 +-sensible (KCa) y sensibles a la tensi&oacute;n (Kv) K + bloqueo de canal y disminuido despu&eacute;s del tratamiento de diltiazem. En PHTN PA SMC, KCa bloqueo no tuvo ning&uacute;n efecto, mientras que aumentan de diltiazem y bloqueo de Kv [Ca2 +] i. inhibici&oacute;n del ret&iacute;culo sarcopl&aacute;smico actividad Ca2 + ATPasa causaron un aumento mayor en la [Ca2 +] i en controles en comparaci&oacute;n con PHTN PA SMC. Por el contrario, rianodina caus&oacute; un mayor aumento de [Ca2 +] i PHTN en comparaci&oacute;n con control SMC PA. Canales de KCa que mRNA se disminuye y Kv que mRNA es sin cambios en PHTN PA SMC comparan con controles. Concluimos que PHTN compromete PA SMC O2 sensor, altera la homeostasis del Ca2 + intracelular y cambia el canal de iones predominantes que determina basal [Ca2 +] i de KCa a Kv.

La hipertensi&oacute;n pulmonar cr&oacute;nica intrauterina compromete la arteria pulmonar fetal m&uacute;sculo liso celular O2 sensor.

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca(2+)](i)) through inactivation of a K(+) channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca(2+)](i) by 11.7 +/- 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca(2+)](i) was mimicked by pharmacological blockade of the voltage-sensitive K(+) channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca(2+)](i). Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca(2+)](i). Endothelin increased [Ca(2+)](i) in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca(2+)](i) via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.

Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores.

At birth, the lung environment changes from low to relatively high O(2) tension. Pulmonary blood flow increases and pulmonary artery pressure decreases. Recent data suggest that pulmonary vascular calcium-sensitive K(+) channel (BK(Ca)) activation mediates perinatal pulmonary vasodilation. Although BK(Ca) channel expression is developmentally regulated, the molecular mechanisms responsible for BK(Ca) expression remain unknown. We tested the hypothesis that the low-O(2) tension environment of the normal fetus modulates BK(Ca) channel expression. We analyzed BK(Ca) expression under conditions of hypoxia and normoxia both in vitro and in vivo. BK(Ca) alpha-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, BK(Ca) expression was similarly affected by hypoxia. When adult Sprague-Dawley rats were placed in hypobaric hypoxic chambers for 3 wk, hypoxic animals showed an increase of threefold in the expression of BK(Ca) alpha- and more than twofold in the expression of BK(Ca) beta(1)-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the beta-subunit of the BK(Ca), both BK(Ca) beta(1)- and beta(2)-subunit luciferase (K(Ca) beta:luc(+)) reporter genes were constructed. Hypoxia increased PASMC K(Ca) beta(1):luc(+) reporter expression by threefold and K(Ca) beta(2):luc(+) expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in BK(Ca) channel alpha- and beta(1)-subunit expression, respectively. Together, these results suggest that oxygen tension modulates BK(Ca) channel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level.

Oxygen tension modulates the expression of pulmonary vascular BKCa channel alpha- and beta-subunits.

Pulmonary artery smooth muscle cell (PASMC) relaxation at birth results from an increase in cytosolic cGMP, cGMP-dependent and kinase-mediated activation of the Ca2+-sensitive K+ channel (KCa), and closure of voltage-operated Ca2+ channels (VOCC). How chronic intrauterine pulmonary hypertension compromises perinatal pulmonary vasodilation remains unknown. We tested the hypothesis that chronic intrauterine pulmonary hypertension selectively modifies gene expression to mitigate perinatal pulmonary vasodilation mediated by the cGMP kinase-KCa-VOCC pathway. PASMC were isolated from late-gestation fetal lambs that had undergone either ligation of the ductus arteriosus (hypertensive) or sham operation (control) at 127 days of gestation and were maintained under either hypoxic (approximately 25 Torr) or normoxic (approximately 120 Torr) conditions in primary culture. We studied mRNA levels for cGMP kinase Ialpha (PKG-1alpha), the alpha-chain of VOCC (Cav1.2), and the alpha-subunit of the KCa channel. Compared with control PASMC, hypertensive PASMC had decreased VOCC, KCa, and PKG-1alpha expression. In response to sustained normoxia, expression of VOCC and KCa channel decreased and expression of PKG-1alpha increased. In contrast, sustained normoxia had no effect on PKG-1alpha levels and an attenuated effect on VOCC and KCa channel expression in hypertensive PASMC. Protein expression of PKG-1alpha was consistent with the mRNA data. We conclude that chronic intrauterine pulmonary hypertension decreases PKG expression and mitigates the genetic effects of sustained normoxia on pulmonary vasodilation, because gene expression remains compromised even after sustained exposure to normoxia.

Chronic intrauterine pulmonary hypertension selectively modifies pulmonary artery smooth muscle cell gene expression.

Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.

Lung specific developmental expression of the Xenopus laevis surfactant protein C and B genes.

To test the hypothesis that an acute increase in O(2) tension increases cytosolic calcium ([Ca(2+)](i)) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (<10 passages) were isolated from the intralobar pulmonary artery (PA) of fetal sheep and maintained under hypoxic conditions (Po(2), 25 Torr). Using the calcium-sensitive dye fura-2, we demonstrated that acute normoxia (Po(2) = 120 Torr) increased PAECs [Ca(2+)](i) by increasing the rate of entry of extracellular calcium. In the presence of either ryanodine or 2-aminoethoxy-diphenylborate (2APB), normoxia did not lead to a sustained increase in PAECs [Ca(2+)](i) Whole-cell patch clamp studies demonstrated that acute normoxia causes PAEC membrane depolarization. When loaded with the nitric oxide (NO)-sensitive dye, DAF - FM, acute normoxia increased PAEC fluorescence. In PAECs derived from fetal lambs with pulmonary hypertension, an acute increase in O(2) tension had no effect on either [Ca(2+)](i) or NO production. Hypoxia increases loading of acetylcholine-sensitive calcium stores, as hypoxia potentiated the response to acetylcholine We conclude that acute normoxia increases [Ca(2+)](i) and NO production in normotensive but not hypertensive fetal PAECs via extracellular calcium entry and calcium release from calcium-sensitive intracellular stores.

Acute normoxia increases fetal pulmonary artery endothelial cell cytosolic Ca2+ via Ca2+-induced Ca2+ release.

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells.

The transcriptional machinery involved in the transition of an infant from intrauterine to air-breathing life is developmentally regulated, as the fetus and adult manifest differential genetic expression. The low oxygen (O(2)) environment of the mammalian fetus and the increase in O(2) tension that occurs at birth may account for the developmentally regulated alterations in gene expression. We tested the hypothesis that hypoxia-inducible factor 1 (HIF-1) expression, an O(2)-sensitive transcription factor, is developmentally regulated. We found that in fetal pulmonary artery (PA) smooth muscle cells (SMC), fetal HIF-1 protein levels were O(2)-insensitive, whereas in adult PA SMC, hypoxia increased HIF-1 protein expression. Surprisingly, hypoxia increased HIF-1 mRNA expression in fetal, but not in adult, PA SMC. HIF-1 degradation and transcriptional activity is contingent on prolyl- and asparagyl-hydroxylases. To determine whether developmental differences in O(2) sensitivity or expression of these enzymes accounts for the divergence of HIF-1 sensitivity between fetus and adult, we studied the expression of the three most well characterized prolyl-hydroxylases, PHD1, PHD2, and PHD3, and the expression of regulators of HIF-1 transcriptional activity, asparagyl-hydroxylase, factor inhibiting HIF, and the oncogenic factor, CITED2 (CREB-binding protein/p300 interacting transactivator with ED-rich tail). We found that, as in the case of HIF-1, these genes are differentially regulated in the fetus, enabling the mammalian fetus to thrive in the low O(2) tension intrauterine environment even while rendering a newborn infant uniquely well adapted to respond to the acute increase in O(2) tension that occurs at birth.

Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells.

The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP) have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. Advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward ChIP assay and analysis methodologies.

Article	Year
Repression of organizer genes in dorsal and ventral Xenopus cells mediated by maternal XTcf3. Development (Cambridge, England)\| PubMed ID: 12163405	2002
Pulmonary vascular K+ channel expression and vasoreactivity in a model of congenital heart disease. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 12388350	2002
Chronic intrauterine pulmonary hypertension compromises fetal pulmonary artery smooth muscle cell O2 sensing. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 12882761	2003
Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 15695541	2005
Oxygen tension modulates the expression of pulmonary vascular BKCa channel alpha- and beta-subunits. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 16284215	2006
Chronic intrauterine pulmonary hypertension selectively modifies pulmonary artery smooth muscle cell gene expression. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 16467248	2006
Lung specific developmental expression of the Xenopus laevis surfactant protein C and B genes. Gene expression patterns : GEP\| PubMed ID: 16798105	2007
Acute normoxia increases fetal pulmonary artery endothelial cell cytosolic Ca2+ via Ca2+-induced Ca2+ release. Pediatric research\| PubMed ID: 16857761	2006
Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 17158601	2007
Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18000055	2007
Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells. BMC biotechnology\| PubMed ID: 19563662	2009

Ernesto Resnik

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