contrast-imaging-in-mouse-embryos-using-high-frequency-ultrasound

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

University of Toronto

Sunnybrook Research Institute

Mount Sinai Hospital, Toronto

The molecular mechanisms responsible for organ-specific differences in vascular development are not well established. Animals lacking the receptor tyrosine kinase Tie1 die of hemorrhage and pulmonary edema. Furthermore, cells lacking Tie1 are excluded from blood vessels of the mature lung. These findings suggest the importance of Tie1 in the pulmonary vasculature. We quantified the organ-specific expression of Tie1 during embryonic and postnatal murine development using both quantitative real-time polymerase chain reaction (PCR) and chemiluminescence employing a tie1.lacZ reporter. In the lung, Tie1 expression increases markedly immediately prior to birth and rises further in the newborn animal, a pattern not found in other organs. Furthermore, expression of Tie1 in the lung is also unique by its persistent increase in the adult animal. This unique pattern of Tie1 gene expression in the embryonic and mature lung supports a distinct role for Tie1 in the development and function of the pulmonary vasculature.

A unique pattern of Tie1 expression in the developing murine lung.

In mammals, the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region, within cells localized to the ventral floor of the major blood vessels, suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells, their precursors, and HCs, suggestive of a role in the divergence and function of both lineages. Indeed, gene targeting approaches have shown that TIE1, TIE2, and ligands for TIE2, the angiopoietins, are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs, we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly, however, these receptors are specifically required during postnatal bone marrow hematopoiesis.

Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis.

&#x5FC3;&#x8840;&#x7BA1;&#x7CFB;&#x7EDF;&#x7684;&#x53D1;&#x5C55;&#x548C;&#x65E9;&#x671F;&#x9020;&#x8840;&#x7CFB;&#x7EDF;&#x7684;&#x53D1;&#x5C55;&#x5BC6;&#x5207;&#x76F8;&#x5173;&#x7684;&#x548C;&#x4E24;&#x8005;&#x90FD;&#x9700;&#x8981;&#x901A;&#x8FC7; Tie2 &#x53D7;&#x4F53;&#x916A;&#x6C28;&#x9178;&#x6FC0;&#x9176;&#x4FE1;&#x53F7;&#x3002;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x548C;&#x9020;&#x8840;&#x7EC6;&#x80DE;&#xFF0C;&#x4EE5;&#x53CA;&#x5176;&#x524D;&#x4F53;&#x5728;&#x5F00;&#x53D1;&#x8FC7;&#x7A0B;&#x4E2D;&#x5171;&#x4EAB;&#x5171;&#x540C;&#x7684;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#xFF0C;&#x5C3D;&#x7BA1;&#x5B83;&#x4ECD;&#x7136;&#x5B8C;&#x5168;&#x672A;&#x77E5;&#x5982;&#x4F55;&#x534F;&#x8C03; Tie2 &#x4FE1;&#x53F7;&#x8C03;&#x63A7;&#x5FC3;&#x8840;&#x7BA1;&#x53D1;&#x80B2;&#x548C;&#x4F53;&#x5185;&#x7684;&#x65E9;&#x671F;&#x9020;&#x8840;&#x529F;&#x80FD;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x8FD9;&#x91CC;&#x5C55;&#x793A; Tie2 &#x916A;&#x6C28;&#x9178;&#x7FA7;&#x57FA;&#x672B;&#x7AEF; 1100&#x5E74;&#x6B8B;&#x7559;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7A81;&#x53D8;&#x5C0F;&#x9F20;&#x663E;&#x793A;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x7F3A;&#x9677;&#x548C;&#x635F;&#x5BB3;&#x9020;&#x8840;&#x548C;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x53D1;&#x80B2;&#x7684;&#x4E2D;&#x80DA;&#x8FDB;&#x5C55; splanchnopleural &#x5C42;&#x7C7B;&#x4F3C; Tie2 null &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5C0F;&#x9F20;&#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x3002;&#x5947;&#x602A;&#x7684;&#x662F;&#xFF0C;&#x4E0D;&#x8FC7;&#xFF0C;&#x4E0E; Tie2 null &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5C0F;&#x9F20;&#x4E0D;&#x540C;&#x5C0F;&#x9F20;&#x4FE1;&#x53F7;&#x901A;&#x8FC7;&#x6B64;&#x916A;&#x6C28;&#x9178;&#x6B8B;&#x7559;&#x6709;&#x4E0D;&#x8DB3;&#x4E4B;&#x5904;&#x662F;&#x663E;&#x793A;&#x8840;&#x7BA1;&#x5468;&#x56F4;&#x7EC6;&#x80DE;&#x4E0E;&#x65B0;&#x751F;&#x8840;&#x7BA1;&#x6B63;&#x5E38;&#x534F;&#x4F1A;&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x662F;&#x7B2C;&#x4E00;&#x6B21;&#x8BC1;&#x660E; Tie2&#xFF0C;&#x5355;&#x4E2A;&#x916A;&#x6C28;&#x9178;&#x6B8B;&#x7559;&#x7684;&#x751F;&#x7406;&#x91CD;&#x8981;&#x6027;&#x548C;&#x4ED6;&#x4EEC;&#x5EFA;&#x8BAE;&#x53D7;&#x4F53;&#x4E2D;&#x7684;&#x591A;&#x4E2A;&#x916A;&#x6C28;&#x9178;&#x6B8B;&#x57FA;&#x53EF;&#x534F;&#x8C03;&#x5FC3;&#x8840;&#x7BA1;&#x53D1;&#x5C55;&#x548C;&#x65E9;&#x671F;&#x9020;&#x8840;&#x7EC6;&#x80DE;&#x53D1;&#x80B2;&#x3002;

&#x72EC;&#x7279;&#x7684;&#x916A;&#x6C28;&#x9178;&#x5BF9; Tie2 &#x4E2D;&#x7684;&#x6B8B;&#x7559;&#x91CF;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x548C;&#x65E9;&#x671F;&#x9020;&#x8840;&#x7684;&#x9009;&#x62E9;&#x6027;&#x4F5C;&#x7528;&#x3002;

The development of the cardiovascular system and the development of the early hematopoietic systems are closely related, and both require signaling through the Tie2 receptor tyrosine kinase. Although endothelial cells and hematopoietic cells as well as their precursors share common gene expression patterns during development, it remains completely unknown how Tie2 signaling coordinately regulates cardiovascular development and early hematopoiesis in vivo. We show here that mice with a targeted mutation in tyrosine residue 1100 in the carboxyl-terminal tail of Tie2 display defective cardiac development and impaired hematopoietic and endothelial cell development in the paraaortic splanchnopleural mesoderm similar to that seen in Tie2-null mutant mice. Surprisingly, however, unlike Tie2-null mutant mice, mice deficient in signaling through this tyrosine residue show a normal association of perivascular cells with nascent blood vessels. These studies are the first to demonstrate the physiological importance of a single tyrosine residue in Tie2, and they suggest that multiple tyrosine residues in the receptor may coordinate cardiovascular development and early hematopoietic development.

Selective role of a distinct tyrosine residue on Tie2 in heart development and early hematopoiesis.

Le d&#xE9;veloppement du syst&#xE8;me cardio-vasculaire et le d&#xE9;veloppement des premiers syst&#xE8;mes h&#xE9;matopo&#xEF;&#xE9;tiques sont &#xE9;troitement li&#xE9;es, et les deux n&#xE9;cessitent la signalisation par le r&#xE9;cepteur &#xE0; activit&#xE9; tyrosine kinase Tie2. Bien que les cellules endoth&#xE9;liales et les cellules h&#xE9;matopo&#xEF;&#xE9;tiques ainsi que de leurs pr&#xE9;curseurs ont en commun l&#x26;#39;expression des g&#xE8;nes au cours du d&#xE9;veloppement, il reste totalement inconnu comment Tie2 signalisation r&#xE9;glemente de mani&#xE8;re coordonn&#xE9;e le d&#xE9;veloppement cardiovasculaire et l&#x26;#39;h&#xE9;matopo&#xEF;&#xE8;se pr&#xE9;coce in vivo. Nous montrons ici que les souris avec une mutation cibl&#xE9;e dans les r&#xE9;sidus de tyrosine 1100 dans la queue carboxy-terminal de Tie2 afficher un d&#xE9;faut de d&#xE9;veloppement cardiaque et des troubles de d&#xE9;veloppement des cellules h&#xE9;matopo&#xEF;&#xE9;tiques et endoth&#xE9;liales dans le m&#xE9;soderme para-aortiques splanchnopleural similaire &#xE0; celle observ&#xE9;e dans Tie2-souris mutantes nulles. &#xC9;tonnamment, toutefois, contrairement &#xE0; Tie2-souris mutantes nulles, les souris d&#xE9;ficientes en signalisation &#xE0; travers ce r&#xE9;sidu tyrosine montrent une association normale des cellules p&#xE9;rivasculaires des vaisseaux sanguins naissants. Ces &#xE9;tudes sont les premiers &#xE0; d&#xE9;montrer l&#x26;#39;importance physiologique d&#x26;#39;un seul r&#xE9;sidu tyrosine dans Tie2, et ils sugg&#xE8;rent que plusieurs r&#xE9;sidus tyrosine du r&#xE9;cepteur peut coordonner le d&#xE9;veloppement cardiovasculaire et au d&#xE9;but du d&#xE9;veloppement h&#xE9;matopo&#xEF;&#xE9;tique.

R&#xF4;le s&#xE9;lective d&#x26;#39;un r&#xE9;sidu tyrosine Distinct sur Tie2 dans le d&#xE9;veloppement du c&#x153;ur et h&#xE9;matopo&#xEF;&#xE8;se pr&#xE9;coce

Selektive Rolle eines gesonderten Tyrosinrest auf Tie2 in die Entwicklung des Herzens und der fr&#xFC;hen H&#xE4;matopoese.

Lo sviluppo del sistema cardiovascolare e lo sviluppo dei primi sistemi ematopoietici sono strettamente correlati, ed entrambi richiedono di segnalazione attraverso la chinasi della tirosina del recettore Tie2. Anche se le cellule endoteliali e cellule ematopoietiche cos&igrave; come i loro precursori condividono comuni pattern di espressione genica durante lo sviluppo, rimane completamente sconosciuta come segnalazione Tie2 coordinately regola lo sviluppo cardiovascolare e primi ematopoiesi in vivo. Mostriamo qui che i topi con una mutazione mirata in 1100 di residui di tirosina nella coda carbossi-terminale di Tie2 display difettoso sviluppo cardiaco e ha alterato lo sviluppo delle cellule ematopoietiche ed endoteliali nel mesoderma splanchnopleural paraaortic simile a quello osservato nei topi mutanti Tie2-null. Sorprendentemente, per&ograve;, a differenza dei topi mutanti Tie2-null, topi carenti di segnalazione attraverso questo residuo di tirosina mostrano una normale associazione di cellule perivascolari con vasi sanguigni nascente. Questi studi sono il primo a dimostrare l&#39;importanza fisiologica di un residuo di tirosina singolo in Tie2, e suggeriscono che pi&ugrave; residui di tirosina nel recettore possono coordinare lo sviluppo cardiovascolare e precoce sviluppo ematopoietico.

Ruolo selettivo di un residuo di tirosina distinti su Tie2 nello sviluppo del cuore e primi ematopoiesi.

&#x5FC3;&#x81D3;&#x8840;&#x7BA1;&#x7CFB;&#x306E;&#x958B;&#x767A;&#x3068;&#x65E9;&#x671F;&#x306E;&#x9020;&#x8840;&#x7CFB;&#x306E;&#x958B;&#x767A;&#x304C;&#x5BC6;&#x63A5;&#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3001;&#x3069;&#x3061;&#x3089;&#x3082;Tie2&#x53D7;&#x5BB9;&#x4F53;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x304C;&#x5FC5;&#x8981;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x304A;&#x3088;&#x3073;&#x9020;&#x8840;&#x7D30;&#x80DE;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x305D;&#x306E;&#x524D;&#x99C6;&#x4F53;&#x304C;&#x958B;&#x767A;&#x4E2D;&#x306B;&#x5171;&#x901A;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x5171;&#x6709;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x304C;&#x3001;&#x305D;&#x308C;&#x304C;Tie2&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x306F;&#x5354;&#x8ABF;&#x7684;&#x306B;in vivo&#x3067;&#x306E;&#x5FC3;&#x8840;&#x7BA1;&#x7CFB;&#x306E;&#x958B;&#x767A;&#x3068;&#x65E9;&#x671F;&#x306E;&#x9020;&#x8840;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x5B8C;&#x5168;&#x306B;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308B;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;Tie2&#x306E;&#x30AB;&#x30EB;&#x30DC;&#x30AD;&#x30B7;&#x30EB;&#x672B;&#x7AEF;&#x5C3E;&#x90E8;&#x306E;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6B8B;&#x57FA;1100&#x3092;&#x5BFE;&#x8C61;&#x3068;&#x5909;&#x7570;&#x3092;&#x6301;&#x3064;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x3001;Tie2&#x306E;&#x975E;&#x30CC;&#x30EB;&#x5909;&#x7570;&#x30DE;&#x30A6;&#x30B9;&#x306B;&#x898B;&#x3089;&#x308C;&#x308B;&#x3082;&#x306E;&#x3068;&#x985E;&#x4F3C;&#x3057;&#x305F;&#x5927;&#x52D5;&#x8108;splanchnopleural&#x4E2D;&#x80DA;&#x8449;&#x306E;&#x6B20;&#x9665;&#x5FC3;&#x81D3;&#x306E;&#x958B;&#x767A;&#x3001;&#x969C;&#x5BB3;&#x3001;&#x9020;&#x8840;&#x304A;&#x3088;&#x3073;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x767A;&#x9054;&#x3092;&#x8868;&#x793A;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x3053;&#x3053;&#x306B;&#x793A;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x9A5A;&#x304F;&#x3079;&#x304D;&#x3053;&#x3068;&#x306B;&#x3001;Tie2&#x306E;&#x975E;&#x30CC;&#x30EB;&#x5909;&#x7570;&#x30DE;&#x30A6;&#x30B9;&#x3068;&#x306F;&#x7570;&#x306A;&#x308A;&#x3001;&#x3053;&#x306E;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6B8B;&#x57FA;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x6B20;&#x640D;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x3001;&#x65B0;&#x751F;&#x8840;&#x7BA1;&#x3068;&#x8840;&#x7BA1;&#x5468;&#x56F2;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x6B63;&#x5E38;&#x306A;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;Tie2&#x306E;&#x5358;&#x4E00;&#x306E;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6B8B;&#x57FA;&#x306E;&#x751F;&#x7406;&#x5B66;&#x7684;&#x91CD;&#x8981;&#x6027;&#x3092;&#x793A;&#x3059;&#x305F;&#x3081;&#x306B;&#x3001;&#x6700;&#x521D;&#x3067;&#x3042;&#x308A;&#x3001;&#x5F7C;&#x3089;&#x306F;&#x53D7;&#x5BB9;&#x4F53;&#x306B;&#x8907;&#x6570;&#x306E;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6B8B;&#x57FA;&#x306F;&#x3001;&#x5FC3;&#x81D3;&#x8840;&#x7BA1;&#x306E;&#x958B;&#x767A;&#x3068;&#x65E9;&#x671F;&#x306E;&#x9020;&#x8840;&#x958B;&#x767A;&#x3092;&#x8ABF;&#x6574;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;&#x3002;

&#x30CF;&#x30FC;&#x30C8;&#x306E;&#x958B;&#x767A;&#x3068;&#x521D;&#x671F;&#x9020;&#x8840;&#x306B;&#x304A;&#x3051;&#x308B;Tie2&#x306E;&#x4E0A;&#x3067;&#x660E;&#x78BA;&#x306A;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6B8B;&#x57FA;&#x306E;&#x9078;&#x629E;&#x7684;&#x5F79;&#x5272;

&#xC2EC;&#xC7A5; &#xD608;&#xAD00; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xAC1C;&#xBC1C; &#xBC0F; &#xC870;&#xAE30; &#xC870; &#xD608; &#xC2DC;&#xC2A4;&#xD15C; &#xAC1C;&#xBC1C; &#xBC00;&#xC811; &#xD558; &#xAC8C; &#xAD00;&#xB828; &#xB41C;, &#xADF8;&#xB9AC;&#xACE0; &#xB450; Tie2 &#xC218;&#xC6A9; &#xCCB4; &#xD2F0;&#xB85C;&#xC2E0; &#xD0A4; &#xB2C8; &#xC544; &#xC81C;&#xB97C; &#xD1B5;&#xD574; &#xC2E0;&#xD638; &#xD544;&#xC694;. &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC640; &#xC870; &#xD608; &#xC138;&#xD3EC; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xADF8;&#xB4E4;&#xC758; &#xC804; &#xB2E8;&#xACC4;&#xC778; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xACF5;&#xD1B5; &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604; &#xD328;&#xD134;&#xC744; &#xACF5;&#xC720;, &#xD558;&#xC9C0;&#xB9CC; &#xB0A8;&#xC544; &#xC644;&#xC804;&#xD788; &#xC54C; &#xC218; &#xC5C6;&#xB294; &#xC5B4;&#xB5BB;&#xAC8C; &#xC2EC;&#xC7A5; &#xD608;&#xAD00; &#xAC1C;&#xBC1C; &#xBC0F; &#xC870;&#xAE30; &#xC870; vivo&#xC5D0;&#xC11C; &#xC870;&#xC808; coordinately Tie2 &#xC2E0;&#xD638;. &#xC5EC;&#xAE30; Tie2 &#xD2F0;&#xB85C;&#xC2E0; &#xC794;&#xB958;&#xBB3C; 1100 carboxyl &#xD130;&#xBBF8;&#xB110; &#xAF2C;&#xB9AC;&#xC5D0; &#xD45C;&#xC801;&#xC73C;&#xB85C; &#xD55C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB9C8;&#xC6B0;&#xC2A4; &#xACB0;&#xD568;&#xC774; &#xC2EC;&#xC7A5; &#xAC1C;&#xBC1C; &#xD45C;&#xC2DC; &#xD558; &#xACE0; &#xC870; &#xD608;&#xACFC; &#xB0B4; &#xD53C; &#xC138;&#xD3EC; &#xAC1C;&#xBC1C; Tie2 null &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC0DD;&#xC950;&#xC5D0;&#xC11C; &#xBCF8; &#xBE44;&#xC2B7;&#xD55C; paraaortic splanchnopleural mesoderm&#xC5D0;&#xC11C; &#xC7A5;&#xC560;&#xC778;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; &#xB180;&#xB78D;&#xAC8C;&#xB3C4;,, Tie2 null &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC950;&#xC640;&#xB294; &#xB2EC;&#xB9AC; &#xB9C8;&#xC6B0;&#xC2A4; &#xACB0;&#xD54D;&#xC774; &#xD2F0;&#xB85C;&#xC2E0; &#xC794;&#xB958;&#xBB3C;&#xC744; &#xD1B5;&#xD574; &#xC2E0;&#xD638;&#xC5D0; &#xD45C;&#xC2DC; &#xCD08;&#xAE30; &#xD608;&#xAD00; perivascular &#xC138;&#xD3EC;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xD611;&#xD68C;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C;&#xB294; &#xBA3C;&#xC800; Tie2&#xC5D0;&#xC11C; &#xB2E8;&#xC77C; &#xD2F0;&#xB85C;&#xC2E0; &#xC794;&#xB958;&#xBB3C;&#xC758; &#xC0DD;&#xB9AC; &#xC801; &#xC911;&#xC694;&#xC131;&#xC744; &#xC785;&#xC99D; &#xD558; &#xACE0; &#xADF8;&#xB4E4;&#xC740; &#xC2EC;&#xC7A5; &#xD608;&#xAD00; &#xAC1C;&#xBC1C; &#xBC0F; &#xCD08;&#xAE30; hematopoietic &#xAC1C;&#xBC1C; &#xC218;&#xC6A9; &#xCCB4;&#xC5D0;&#xC11C; &#xC5EC;&#xB7EC; &#xD2F0;&#xB85C;&#xC2E0; &#xC794;&#xB958;&#xBB3C; &#xC870;&#xC815; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC2EC;&#xC7A5; &#xBC1C;&#xB2EC; &#xBC0F; &#xC870;&#xAE30; &#xC870; tie2&#xC5D0; &#xB69C;&#xB837;&#xD55C; &#xD2F0;&#xB85C;&#xC2E0; &#xC794;&#xB958;&#xBB3C;&#xC758; &#xC120;&#xD0DD;&#xC801; &#xC5ED;&#xD560;.

El desarrollo del sistema cardiovascular y el desarrollo de los sistemas hematopoy&#xE9;ticos tempranos est&#xE1;n estrechamente relacionados, y ambos requieren de se&#xF1;alizaci&#xF3;n a trav&#xE9;s de la tirosina quinasa del receptor Tie2. Aunque las c&#xE9;lulas endoteliales y las c&#xE9;lulas hematopoy&#xE9;ticas, as&#xED; como sus precursores compartir comunes patrones de expresi&#xF3;n g&#xE9;nica durante el desarrollo, sigue siendo totalmente desconocido c&#xF3;mo Tie2 se&#xF1;alizaci&#xF3;n coordinadamente regula el desarrollo cardiovascular y hematopoyesis temprana in vivo. Mostramos aqu&#xED; que los ratones con una mutaci&#xF3;n espec&#xED;fica en el residuo de tirosina 1100 en la cola carboxilo-terminal de Tie2 mostrar defectos en el desarrollo card&#xED;aco y problemas de desarrollo de c&#xE9;lulas hematopoy&#xE9;ticas y c&#xE9;lulas endoteliales en el mesodermo paraa&#xF3;rtica splanchnopleural similar a la observada en Tie2 nulo ratones mutantes. Sorprendentemente, sin embargo, a diferencia de Tie2 nulo ratones mutantes, los ratones deficientes en la se&#xF1;alizaci&#xF3;n a trav&#xE9;s de este residuo de tirosina muestran una asociaci&#xF3;n normal de las c&#xE9;lulas perivasculares en los vasos sangu&#xED;neos nacientes. Estos estudios son los primeros en demostrar la importancia fisiol&#xF3;gica de un &#xFA;nico residuo de tirosina en Tie2, y sugieren que varios residuos de tirosina en el receptor puede coordinar el desarrollo cardiovascular y el desarrollo hematopoy&#xE9;tico temprano.

Funci&#xF3;n selectiva de un residuo de tirosina distinto en Tie2 en el desarrollo del coraz&#xF3;n y la hematopoyesis temprana

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-beta superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo.

Extraordinary advances in pluripotent stem cell research have initiated an era of hope for regenerative strategies to treat human disease. Besides embryonic stem cells, the discovery of induced pluripotent stem cells widened the possibility of patient-specific cell therapy, drug discovery, and disease modeling. Although similar, it has become clear that these two pluripotent cell types display significant differences. In this review, we explore current knowledge of the molecular and functional similarities and differences between these two cell types to emphasize the necessity for thorough characterization of their properties as well as their differentiation capabilities in the pluripotent state. Such comparative studies will be crucial for determining the more suitable cell type for future stem cell-based therapies for human degenerative diseases.

Concise review: Embryonic stem cells versus induced pluripotent stem cells: the game is on.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.

As a tumor surrogate, the mouse embryo presents as an excellent alternative for examining the binding of angiogenesis-targeting microbubbles and assessing the quantitative nature of molecular ultrasound. We establish the validity of this model by developing a robust method to study microbubble kinetic behavior and investigate the reproducibility of targeted binding in the murine embryo. Vascular endothelial growth factor receptor 2 (VEGFR2)-targeted (MBV), rat immunoglobulin G2 (IgG2) control antibody-targeted (MBC) and untargeted (MBU) microbubbles were introduced into vasculature of living mouse embryos. Non-linear contrast-specific and B-mode ultrasound imaging, performed at 21 MHz with a Vevo-2100 scanner, was used to collect basic perfusion parameters and contrast mean power ratios for all bubble types. We observed a twofold increase (p < 0.001) in contrast mean power ratios for MBV (4.14 ± 1.78) compared with those for MBC (1.95 ± 0.78) and MBU (1.79 ± 0.45). Targeted imaging of endogenous endothelial cell surface markers in mouse embryos is possible with labeled microbubbles. The mouse embryo thus presents as a versatile model for testing the performance of ultrasound molecular targeting, where further development of quantitative imaging techniques may enable rapid evaluations of biomarker expression in studies of vascular development, disease and angiogenesis.

VEGFR2-targeted molecular imaging in the mouse embryo: an alternative to the tumor model.

Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

An epigenomic roadmap to induced pluripotency reveals DNA methylation as a reprogramming modulator.

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.

Small RNA changes en route to distinct cellular states of induced pluripotency.

The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated -omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Divergent reprogramming routes lead to alternative stem-cell states.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

Genome-wide characterization of the routes to pluripotency.

Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality around embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and Angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling and EnMT during cardiovascular development.

Article	Year
A unique pattern of Tie1 expression in the developing murine lung. Experimental lung research\| PubMed ID: 12554357	2003
Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 14530387	2003
Selective role of a distinct tyrosine residue on Tie2 in heart development and early hematopoiesis. Molecular and cellular biology\| PubMed ID: 15899871	2005
Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo. Developmental biology\| PubMed ID: 19703439	2009
Concise review: Embryonic stem cells versus induced pluripotent stem cells: the game is on. Stem cells (Dayton, Ohio)\| PubMed ID: 22102565	2012
Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22509029	2012
VEGFR2-targeted molecular imaging in the mouse embryo: an alternative to the tumor model. Ultrasound in medicine & biology\| PubMed ID: 24342913	2014
An epigenomic roadmap to induced pluripotency reveals DNA methylation as a reprogramming modulator. Nature communications\| PubMed ID: 25493341	2014
Small RNA changes en route to distinct cellular states of induced pluripotency. Nature communications\| PubMed ID: 25494340	2014
Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks. Nature communications\| PubMed ID: 25494451	2014
Divergent reprogramming routes lead to alternative stem-cell states. Nature\| PubMed ID: 25503232	2014
Genome-wide characterization of the routes to pluripotency. Nature\| PubMed ID: 25503233	2014
Requisite role for Nck adaptors in cardiovascular development, endothelial-to mesenchymal transition and directed cell migration. Molecular and cellular biology\| PubMed ID: 25691664	2015

Mira C. Puri

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