High Resolution Fluorescent <em>In Situ</em> Hybridization in <em>Drosophila</em> Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

University of Toronto

2 &#x3064;&#x306E;&#x521D;&#x671F;&#x80DA;&#x80DA;&#x30D5;&#x30A9;&#x30FC;&#x30E0; &#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x958B;&#x767A;&#x306E;&#x65E9;&#x3044;&#x6BB5;&#x968E;&#x3067;&#x306F;&#x3002;1 &#x3064;&#x306F;&#x3001;amnioserosa&#x3001;&#x80DA;&#x82BD;&#x30D0;&#x30F3;&#x30C9;&#x64A4;&#x56DE;&#x3068;&#x80CC;&#x5074;&#x306E;&#x9589;&#x9396;&#x306E;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x4ED6;&#x3001;&#x5375;&#x9EC4;&#x56A2;&#x306E;&#x767A;&#x9054;&#x306E;&#x5F79;&#x5272;&#x306F;&#x3042;&#x3044;&#x307E;&#x3044;&#x3067;&#x3059;&#x3002;

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Drosophila &#xAC1C;&#xBC1C; &#xCD08;&#xAE30;&#xC5D0; &#xB450; extraembryonic &#xC870;&#xC9C1; &#xD615;&#xD0DC;&#xC785;&#xB2C8;&#xB2E4;. &#xD558;&#xB098;, amnioserosa, &#xC138;&#xADE0; &#xBC34;&#xB4DC; &#xCCA0;&#xD68C;&#xC640; &#xC9C0; &#xD3D0;&#xC1C4;&#xC758; &#xC804;&#xCCB4;&#xC801; &#xACFC;&#xC815;&#xC5D0; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4; &#xC788;&#xB2E4;. &#xB178; &#xB978; &#xC790; sac &#xB2E4;&#xB978; &#xAC1C;&#xBC1C; &#xC5ED;&#xD560;&#xC740; &#xC560;&#xB9E4; &#xD55C;.

Drosophila extraembryonic &#xB9C9; Integrin &#xC885;&#xC18D; apposition morphogenesis &#xC62C;&#xB9AC;&#xACE0; anoikis &#xBC29;&#xC9C0; &#xB429;&#xB2C8;&#xB2E4;.

Forma de dos tejidos extraembrionarias temprano en el desarrollo de Drosophila. Uno, la amnioserosa, se ha implicado en los procesos morfogen&eacute;ticos de retracci&oacute;n de la banda del germen y cierre dorsal. El papel del desarrollo de la otra, el saco vitelino, es obscuro.

Aposici&oacute;n de la integrina-dependiente de Membranas Extraembrionarias Drosophila promueve la morfog&eacute;nesis y previene la anoikis.

&#x4E24;&#x4E2A;&#x80DA;&#x5916;&#x7EC4;&#x7EC7;&#x5728;&#x679C;&#x8747;&#x5F00;&#x53D1;&#x7684;&#x65E9;&#x671F;&#x5F62;&#x5F0F;&#x3002;&#x5176;&#x4E2D;&#x4E00;&#x4E2A;&#xFF0C;amnioserosa&#xFF0C;&#x6709;&#x7275;&#x8FDE;&#x80DA;&#x82BD;&#x4E50;&#x961F;&#x9000;&#x7F29;&#x548C;&#x80CC;&#x95ED;&#x5408;&#x7684;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x8FC7;&#x7A0B;&#x4E2D;&#x3002;&#x53E6;&#xFF0C;&#x5375;&#x9EC4;&#x56CA;&#x4E2D;&#x7684;&#x53D1;&#x5C55;&#x4F5C;&#x7528;&#x662F;&#x6666;&#x6DA9;&#x3002;

&#x6574;&#x5408;&#x7D20;&#x4F9D;&#x8D56;&#x4F59;&#x534E;&#x7684;&#x679C;&#x8747;&#x80DA;&#x5916;&#x819C;&#x4FC3;&#x8FDB;&#x7684;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x548C;&#x9632;&#x6B62;&#x8131;&#x843D;&#x51CB;&#x4EA1;&#x3002;

Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure.

Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis.

Forme de deux tissus extra-embryonnaires t&ocirc;t dans le d&eacute;veloppement de la drosophile. L&#39;un d&#39;entre eux, l&#39;amnioserosa, a &eacute;t&eacute; impliqu&eacute; dans les processus morphog&eacute;n&eacute;tiques de r&eacute;traction bande germe et fermeture dorsale. Le r&ocirc;le du d&eacute;veloppement de l&#39;autre, le sac vitellin, est obscur.

D&eacute;pendant de l&#39;int&eacute;grine apposition des membranes extraembryonnaires Drosophila favorise la morphogen&egrave;se et emp&ecirc;che l&#39;anoikis.

Integrin-abh&#xE4;ngige Anlagerung von Drosophila extraembryonalen Membranen f&#xF6;rdert Morphogenese und verhindert Anoikis.

Forma di due tessuti extraembryonic presto nello sviluppo di Drosophila. Uno, il amnioserosa, &egrave; stato implicato nei processi morfogenetici del germe band retrazione e chiusura dorsale. Il ruolo dello sviluppo di altro, il sacco vitellino, &egrave; oscuro.

Apposizione di integrina-dipendente delle membrane extraembryonic drosofila promuove la morfogenesi e impedisce l&#39;anoikis.

&#x7F16;&#x7801;&#x7531;&#x4E8B;&#x540E; &#xFF08;&#x9AD8;&#x65B0;&#x6280;&#x672F;&#xFF09; &#x8F68;&#x8FF9;&#x7684;&#x6838;&#x950C;&#x6307;&#x86CB;&#x767D;&#x8C03;&#x8282;&#x679C;&#x8747;&#x7684;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#xFF0C;&#x5305;&#x62EC;&#x541B; N-&#x672B;&#x7AEF;&#x6FC0;&#x9176; (JNK) &#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x4E0E;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x7684;&#x805A;&#x5408;&#x51E0;&#x4E2A;&#x7EC6;&#x80DE;&#x8FC7;&#x7A0B;&#x3002;&#x8FD9;&#x4E9B;&#x5206;&#x5B50;&#x7684;&#x8DEF;&#x5F84;&#x4E2D;&#x7684;&#x7F3A;&#x9677;&#x53EF;&#x80FD;&#x662F;&#x5F02;&#x5E38;&#x7EC6;&#x80DE;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#xFF0C;&#x635F;&#x5931;&#x7684;&#x4E0A;&#x76AE;&#x5B8C;&#x6574;&#x6027;&#x548C;&#x51CB;&#x4EA1;&#x53D1;&#x751F;&#x7A81;&#x53D8;&#x4F53;&#x9AD8;&#x65B0;&#x6280;&#x672F;&#xFF0C;&#x53CD;&#x8FC7;&#x6765;&#x5BFC;&#x81F4;&#x5931;&#x8D25;&#x7684;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x8FC7;&#x7A0B;&#xFF0C;&#x5982;&#x80DA;&#x82BD;&#x4E50;&#x961F;&#x9000;&#x7F29;&#x548C;&#x80DA;&#x80CC;&#x95ED;&#x5408;&#x7684;&#x4F9D;&#x636E;&#x3002;&#x82E5;&#x8981;&#x5B9A;&#x4E49;&#x53D7;&#x9AD8;&#x65B0;&#x6280;&#x672F;&#x7684;&#x9057;&#x4F20;&#x9014;&#x5F84;&#xFF0C;124 &#x7B2C;&#x4E8C;&#x548C;&#x7B2C;&#x4E09;&#x4E2A;&#x67D3;&#x8272;&#x4F53;&#x4E0A;&#x7684;&#x4E0D;&#x8DB3;&#x4E4B;&#x5904;&#x548C;&#x7B2C;&#x4E8C;&#x53F7;&#x67D3;&#x8272;&#x4F53;&#x4E0A;&#x7684; 14 &#x91CD;&#x590D;&#x6D4B;&#x5B9A;&#x5242;&#x91CF;&#x654F;&#x611F;&#x4FEE;&#x6539;&#x5F15;&#x8D77;&#x7684;&#x53EF;&#x884C;&#x7684;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#xFF0C;hntpeb &#xFF1B; &#x6E29;&#x5EA6;&#x654F;&#x611F;&#x7C97;&#x7CD9;&#x773C;&#x8868;&#x578B;&#x7684;&#x786E;&#x5B9A;&#x4E86; 29 &#x4E92;&#x52A8;&#x533A;&#x57DF;&#x3002;&#x968F;&#x540E;&#xFF0C;438 P &#x5143;&#x7D20;&#x5F15;&#x8D77;&#x81F4;&#x547D;&#x6027;&#x7A81;&#x53D8;&#x5C06;&#x6620;&#x5C04;&#x5230;&#x8FD9;&#x4E9B;&#x5730;&#x533A;&#x548C; 12 &#x5019;&#x9009;&#x57FA;&#x56E0;&#x9057;&#x4F20;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#xFF0C;&#x5BFC;&#x81F4; 63 &#x663E;&#x6027;&#x4FEE;&#x9970;&#x7B26;&#x5EA7;&#x4F4D;&#x7684;&#x6807;&#x8BC6;&#x8FDB;&#x884C;&#x4E86;&#x6D4B;&#x8BD5;&#x3002;&#x786E;&#x5B9A;&#x7A81;&#x53D8;&#x4F53;&#x7684;&#x4E00;&#x4E2A;&#x5B50;&#x96C6;&#x4E5F;&#x4E3B;&#x8981;&#x4FEE;&#x6539; hnt308 &#x8BF1;&#x5BFC;&#x80DA;&#x80CE;&#x81F4;&#x6B7B;&#x7387;&#xFF0C;&#x56E0;&#x6B64;&#x4EE3;&#x8868;&#x4E00;&#x822C;&#x800C;&#x4E0D;&#x662F;&#x7279;&#x5B9A;&#x4E8E;&#x7EC4;&#x7EC7;&#x7684;&#x6F20;&#x89C6;&#x3002;&#x4E00;&#x822C;&#x975E;&#x540C;&#x5C0F;&#x53EF;&#x5305;&#x62EC;&#x7F16;&#x7801;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x3001; &#x808C;&#x52A8;&#x86CB;&#x767D;&#x7ED3;&#x5408;&#x86CB;&#x767D;&#x3001; &#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x86CB;&#x767D;&#x548C;&#x7EC6;&#x80DE;&#x5916;&#x57FA;&#x8D28;&#x6210;&#x5206;&#x7684;&#x57FA;&#x56E0;&#x4F4D;&#x70B9;&#x3002;&#x51E0;&#x4E2A;&#x975E;&#x540C;&#x5C0F;&#x53EF;&#x7684;&#x8868;&#x8FBE;&#x9AD8;&#x65B0;&#x6280;&#x672F;&#x7A81;&#x53D8;&#x4F53;&#x7EC4;&#x7EC7;&#x4E2D;&#x8FDB;&#x884C;&#x4E86;&#x8BC4;&#x4F30;&#x3002;&#x4E94;&#x4E2A;&#x57FA;&#x56E0; &mdash; &mdash; apontic &#xFF08;apt&#xFF09;&#x3001; &#x4E09;&#x89D2;&#x6D32; (Dl)&#x3001; decapentaplegic (&#x6C11;&#x8FDB;&#x515A;)&#x3001; &#x5CA9;&#x6EB6; &#xFF08;kst&#xFF09; &#x548C;&#x6298;&#x53E0; &#xFF08;&#x4E34;&#x5E02;&#x5C40;&#xFF09; &mdash; &mdash; &#x81EA;&#x4E3B;&#x662F;&#x53D7;&#x89C4;&#x7BA1;&#x7684;&#x7EC4;&#x7EC7;&#xFF0C;&#x56E0;&#x6B64;&#xFF0C;&#x53EF;&#x80FD;&#x76F4;&#x63A5;&#x8F6C;&#x5F55;&#x76EE;&#x6807;&#x7684;&#x9AD8;&#x65B0;&#x6280;&#x672F;&#x3002;&#x4E09;&#x79CD;&#x57FA;&#x56E0;&#x7684; &mdash; &mdash; apt&#x3001; Dl &#x548C;&#x6C11;&#x8FDB;&#x515A; &mdash; &mdash; &#x8FD8;&#x53D7; nonautonomously &#x76F8;&#x90BB;&#x975E;&#x9AD8;&#x65B0;&#x6280;&#x672F;&#x8868;&#x793A;&#x7EC4;&#x7EC7;&#x4E2D;&#x3002;&#x8868;&#x8FBE;&#x7684;&#x51E0;&#x4E2A;&#x9644;&#x52A0;&#x6F20;&#x89C6; &mdash; &mdash; &#x7EF4;&#x4EAC; (vkg)&#x3001; Cg25 &#x548C;&#x5C42;&#x7C98;&#x8FDE;&#x86CB;&#x767D;-alpha (LanA)-&#x4EC5;&#x5728;&#x975E;&#x81EA;&#x6CBB;&#x7684;&#x65B9;&#x5F0F;&#x4F1A;&#x53D7;&#x5230;&#x5F71;&#x54CD;&#x3002;

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The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hntpeb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt308-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes--apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc)--are regulated tissue autonomously and, thus, may be direct transcriptional targets of HNT. Three of these genes--apt, Dl, and dpp--are also regulated nonautonomously in adjacent non-HNT-expressing tissues. The expression of several additional interactors--viking (vkg), Cg25, and laminin-alpha (LanA)-is affected only in a nonautonomous manner.

Dose-sensitive autosomal modifiers identify candidate genes for tissue autonomous and tissue nonautonomous regulation by the Drosophila nuclear zinc-finger protein, hindsight.

La prot&eacute;ine de doigt de zinc nucl&eacute;aire cod&eacute;e par le locus de recul (hnt) r&eacute;glemente plusieurs processus cellulaires dans l&#39;&eacute;pith&eacute;lium de la drosophile, y compris la Jun N-terminale kinase (JNK) polym&eacute;risation de l&#39;actine et de la voie de signalisation. D&eacute;fauts dans ces voies mol&eacute;culaires peuvent sous-tendre les interactions cellulaires anormales, perte de l&#39;int&eacute;grit&eacute; &eacute;pith&eacute;liale et l&#39;apoptose survenant chez les mutants hnt, causant &agrave; son tour &eacute;chec de processus morphog&eacute;n&eacute;tiques comme r&eacute;traction bande germe et fermeture dorsale dans l&#39;embryon. Pour d&eacute;finir les voies g&eacute;n&eacute;tiques r&eacute;glement&eacute;s par hnt, 124 carences sur le deuxi&egrave;me et troisi&egrave;me chromosomes et des duplications sur le second chromosome 14 ont &eacute;t&eacute; test&eacute;es pour modification sensible &agrave; la dose d&#39;un ph&eacute;notype thermosensibles rugueux oeil caus&eacute;e par l&#39;all&egrave;le viable, hntpeb ; 29 r&eacute;gions d&#39;interactions ont &eacute;t&eacute; identifi&eacute;es. Par la suite, des mutations l&eacute;tales induite P 438-&eacute;l&eacute;ment mapping pour ces r&eacute;gions et les g&egrave;nes candidats 12 ont &eacute;t&eacute; test&eacute;es pour interaction g&eacute;n&eacute;tique, conduisant &agrave; l&#39;identification des 63 locus modificateur dominant. Un sous-ensemble des mutants identifi&eacute;s aussi surtout modifier l&eacute;talit&eacute; embryonnaire induite par les hnt308 et repr&eacute;sentent donc g&eacute;n&eacute;ral plut&ocirc;t que des Interactiens de tissu-sp&eacute;cifique. Les Interactiens g&eacute;n&eacute;rales comprennent locus codant des facteurs de transcription, les prot&eacute;ines liant l&#39;actine, prot&eacute;ines de transduction du signal et composants de la matrice extracellulaire. Expression d&#39;interacteurs plusieurs a &eacute;t&eacute; &eacute;valu&eacute;e dans les tissus mutant hnt. Cinq g&egrave;nes--apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), pliss&eacute; (puc)--sont r&eacute;glement&eacute;e tissu autonome et, ainsi, peuvent &ecirc;tre des cibles transcriptionnelles directes de HNT. Trois de ces g&egrave;nes--apt, Dl et dpp--sont &eacute;galement r&eacute;glement&eacute;s nonautonomously dans les tissus adjacents HNT-expression. L&#39;expression de plusieurs Interactiens suppl&eacute;mentaires--viking (vkg) Cg25 et laminine-alpha (LanA)-d&eacute;pend uniquement d&#39;une mani&egrave;re non autonomes.

Dose sensible autosomiques modificateurs identifient les g&egrave;nes candidats de tissu autonome et de r&eacute;gulation autonomes de tissu par la prot&eacute;ine nucl&eacute;aire de doigt de zinc de drosophile, avec le recul.

Dosis-sensitive autosomal-Modifikatoren identifizieren Kandidatengene f&#xFC;r autonome Gewebe und Gewebe nichtautonomen Regulierung durch die nukleare Drosophila Zinkfinger-Protein, im Nachhinein.

La proteina nucleare zinc-finger codificata dal locus senno di poi (hnt) regola diversi processi cellulari in epiteli di drosofila, tra cui la chinasi Jun N-terminal (JNK) signaling pathway e actina polimerizzazione. Difetti in questi percorsi molecolari possono essere alla base delle interazioni cellulari anormali, perdita dell&#39;integrit&agrave; epiteliale e apoptosi che si verifica in hnt mutanti, che a sua volta causa fallimento dei processi morfogenetici come germe band retrazione e chiusura dorsale nell&#39;embrione. Per definire i percorsi genetici regolamentati da hnt, 124 carenze al secondo e terzi cromosomi e 14 duplicazioni sul secondo cromosoma sono stati saggiati per la modifica della dose-dipendente di un fenotipo sensibile alla temperatura massima occhio causato dall&#39;allele vitale, hntpeb; sono stati identificati 29 regioni interagenti. Successivamente, 438 P-elemento-indotta di mutazioni letali mappatura di queste regioni e geni candidati 12 sono state testate per l&#39;interazione genetica, che porta all&#39;identificazione di 63 loci modificatore dominante. Un sottoinsieme dei mutanti identificati anche dominante modificare hnt308-indotta di letalit&agrave; embrionale e rappresentano quindi generale piuttosto che gli Interactiani tessuto-specifici. Interactiani generali includono loci codifica fattori di trascrizione, proteine actina-associazione, proteine di trasduzione del segnale e componenti della matrice extracellulare. Espressione di diversi Interactiani &egrave; stata valutata in tessuto mutante hnt. Cinque geni - apontic (apt), Delta (Dl), decapentaplegic (dpp), Carso (kst) e grinzoso (puc) - sono regolamentato tessuto autonomamente e, quindi, pu&ograve; essere diretti destinazioni transcriptional di HNT. Tre di questi geni - apt, Dl e dpp - sono anche regolati nonautonomously nei tessuti adiacenti non-HNT-esprimere. L&#39;espressione di diversi Interactiani supplementari - viking (vkg), Cg25 e laminina-alfa (LanA)-solo in un modo ne risente.

Dose-dipendente autosomiche modificatori identificano geni candidati per regolamento tessuto e tessuto autonomo dalla proteina zinc-finger nucleare della drosofila, senno di poi.

&#x5F8C;&#x77E5;&#x6075; &#xFF08;&#x96E2;&#x5A5A;&#xFF09; &#x8ECC;&#x8DE1;&#x306B;&#x3088;&#x3063;&#x3066;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C9;&#x3055;&#x308C;&#x305F;&#x6838;&#x4E9C;&#x925B;&#x30D5;&#x30A3;&#x30F3;&#x30AC;&#x30FC;&#x86CB;&#x767D;&#x8CEA;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x7D4C;&#x8DEF;&#x3068;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x306E;&#x91CD;&#x5408; Jun N &#x672B;&#x7AEF;&#x30AD;&#x30CA;&#x30FC;&#x30BC; (JNK) &#x3092;&#x542B;&#x3080;&#x3001;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x7D30;&#x80DE;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x5206;&#x5B50;&#x7D4C;&#x8DEF;&#x306E;&#x6B20;&#x9665;&#x7570;&#x5E38;&#x7D30;&#x80DE;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3001;&#x4E0A;&#x76AE;&#x306E;&#x6574;&#x5408;&#x6027;&#x3001;&#x304A;&#x3088;&#x3073;&#x96E2;&#x5A5A;&#x5909;&#x7570;&#x4F53;&#x3067;&#x767A;&#x751F;&#x3059;&#x308B;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x80DA;&#x82BD;&#x30D0;&#x30F3;&#x30C9;&#x64A4;&#x56DE;&#x3068;&#x80DA;&#x306E;&#x80CC;&#x5074;&#x306E;&#x9589;&#x9396;&#x306A;&#x3069;&#x306E;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x9806;&#x756A;&#x306B;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x969C;&#x5BB3;&#x306E;&#x640D;&#x5931;&#x306E;&#x6839;&#x5E95;&#x306B;&#x3042;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x96E2;&#x5A5A;&#x306B;&#x3088;&#x3063;&#x3066;&#x898F;&#x5236;&#x3055;&#x308C;&#x3066;&#x907A;&#x4F1D;&#x7D4C;&#x8DEF;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;124 &#x306E;&#x4E0D;&#x8DB3;&#x3067;&#x3001;2 &#x756A;&#x76EE;&#x3068; 3 &#x756A;&#x76EE;&#x306E;&#x67D3;&#x8272;&#x4F53;&#x3068; 2 &#x756A;&#x76EE;&#x306E;&#x67D3;&#x8272;&#x4F53;&#x4E0A;&#x306E; 14 &#x306E;&#x91CD;&#x8907;&#x3092;&#x6E2C;&#x5B9A;&#x3057;&#x305F;&#x7DDA;&#x91CF;&#x306B;&#x654F;&#x611F;&#x306A;&#x5909;&#x66F4;&#x306F;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306E;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50; hntpeb &#x306B;&#x3088;&#x3063;&#x3066;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3055;&#x308C;&#x308B;&#x6E29;&#x5EA6;&#x611F;&#x53D7;&#x6027;&#x8352;&#x3044;&#x76EE;&#x306E;&#x8868;&#x73FE;&#x578B;&#x306E;&#x305F;&#x3081;;29 &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x9818;&#x57DF;&#x3092;&#x540C;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305D;&#x306E;&#x5F8C;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E; 12 &#x306E;&#x5019;&#x88DC;&#x907A;&#x4F1D;&#x5B50;&#x304A;&#x3088;&#x3073;&#x9818;&#x57DF;&#x306B;&#x30DE;&#x30C3;&#x30D4;&#x30F3;&#x30B0; 438 P &#x8981;&#x7D20;&#x306B;&#x3088;&#x308B;&#x81F4;&#x6B7B;&#x7A81;&#x7136;&#x5909;&#x7570;&#x306E;&#x907A;&#x4F1D;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3001;63 &#x306E;&#x652F;&#x914D;&#x7684;&#x306A;&#x4FEE;&#x98FE;&#x5B50;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x306E;&#x540C;&#x5B9A;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x30C6;&#x30B9;&#x30C8;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F;&#x7A81;&#x7136;&#x5909;&#x7570;&#x4F53;&#x306E;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x3082;&#x512A;&#x6027; hnt308 &#x8A98;&#x8D77;&#x80DA;&#x6027;&#x81F4;&#x6B7B;&#x3092;&#x5909;&#x66F4;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x7D44;&#x7E54;&#x7279;&#x7570;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x3067;&#x306F;&#x306A;&#x304F;&#x4E00;&#x822C;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E00;&#x822C;&#x7684;&#x306A;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x306B;&#x306F;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x3001;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x7D50;&#x5408;&#x86CB;&#x767D;&#x8CEA;, &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x7D30;&#x80DE;&#x5916;&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306E;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x306B;&#x306F;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002;&#x5F0F;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x96E2;&#x5A5A;&#x30DF;&#x30E5;&#x30FC;&#x30BF;&#x30F3;&#x30C8;&#x7D44;&#x7E54;&#x3067;&#x8A55;&#x4FA1;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;5 &#x3064;&#x306E;&#x907A;&#x4F1D;&#x5B50; &#xFF0D; apontic (apt)&#x3001;&#x30C7;&#x30EB;&#x30BF; (Dl)&#x3001;decapentaplegic (dpp)&#x3001;&#x30AB;&#x30EB;&#x30B9;&#x30C8; &#xFF08;kst&#xFF09;&#x3001;&#x895E; &#xFF08;puc&#xFF09;--&#x898F;&#x5236;&#x7D44;&#x7E54;&#x81EA;&#x5F8B;&#x7684;&#x304A;&#x3088;&#x3073;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x96E2;&#x5A5A;&#x306E;&#x76F4;&#x63A5;&#x8EE2;&#x5199;&#x5236;&#x5FA1;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;3 &#x3064;&#x3053;&#x308C;&#x3089;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306F;--apt &#x306E; Dl &#x3068; dpp--&#x307E;&#x305F; nonautonomously &#x306B;&#x96A3;&#x63A5;&#x3059;&#x308B;&#x96E2;&#x5A5A;-&#x3092;&#x8868;&#x73FE;&#x3059;&#x308B;&#x7D44;&#x7E54;&#x304C;&#x898F;&#x5236;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x8FFD;&#x52A0;&#x306E;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30A2;&#x30AF;&#x30BF;&#x30FC;--&#x30D0;&#x30A4;&#x30AD;&#x30F3;&#x30B0; (vkg)&#x3001;Cg25&#x3001;&#x304A;&#x3088;&#x3073;&#x30E9;&#x30DF;&#x30CB;&#x30F3; &alpha; (LanA) &#x306E;&#x767A;&#x73FE;-&#x81EA;&#x5F8B;&#x7684;&#x306B;&#x306E;&#x307F;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x307E;&#x3059;&#x3002;

&#x7DDA;&#x91CF;&#x306B;&#x654F;&#x611F;&#x306A;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x512A;&#x6027;&#x4FEE;&#x98FE;&#x5B50;&#x81EA;&#x5F8B;&#x578B;&#x7D44;&#x7E54;&#x3068;&#x7D44;&#x7E54;&#x306E;&#x81EA;&#x5F8B;&#x5236;&#x5FA1;&#x306E;&#x305F;&#x3081;&#x306E;&#x5019;&#x88DC;&#x8005;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x5F8C;&#x77E5;&#x6075;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x6838;&#x4E9C;&#x925B;&#x30D5;&#x30A3;&#x30F3;&#x30AC;&#x30FC;&#x86CB;&#x767D;&#x8CEA;&#x306B;&#x3088;&#x308B;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;

&#xB3CC;&#xC774;&#xCF1C;&#xBCF4;&#xBA74; (hnt) &#xB85C;&#xCEE4; &#xC2A4; &#xC758;&#xD574; &#xD575; &#xC544;&#xC5F0; &#xC190;&#xAC00;&#xB77D; &#xB2E8;&#xBC31;&#xC9C8; &#xC870;&#xC808; &#xC2E0;&#xD638; &#xD1B5;&#xB85C; &#xB9D0;&#xB77C; &#xD569; 6 &#xC6D4; N &#xB9E8;&#xB05D; &#xD0A4; (JNK)&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xCD08;&#xD30C;&#xB9AC; &#xC0C1;&#xD53C;&#xC5D0; &#xC5EC;&#xB7EC; &#xC138;&#xD3EC; &#xD504;&#xB85C;&#xC138;&#xC2A4;. &#xC774;&#xB7EC;&#xD55C; &#xBD84;&#xC790; &#xACBD;&#xB85C; &#xBE44;&#xC815;&#xC0C1;&#xC801;&#xC778; &#xC138;&#xD3EC; &#xC0C1;&#xD638; &#xC791;&#xC6A9;, &#xC0C1;&#xD53C; &#xC131;&#xC2E4;&#xACFC; apoptosis hnt &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xBC1C;&#xC0DD; morphogenetic &#xD504;&#xB85C;&#xC138;&#xC2A4; &#xC138;&#xADE0; &#xBC34;&#xB4DC; &#xCCA0;&#xD68C;&#xC640; &#xD0DC;&#xC544; &#xB4F1; &#xD3D0;&#xC1C4; &#xB4F1;&#xC758; &#xCC28;&#xB840;&#xB85C; &#xC77C;&#xC73C;&#xD0A4;&#xB294; &#xC2E4;&#xD328;&#xC758; &#xC190;&#xC2E4; &#xAE30;&#xCD08;&#xAC00; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. Hnt&#xC5D0; &#xC758;&#xD574; &#xADDC;&#xC81C; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xACBD;&#xB85C; &#xC815;&#xC758; &#xD558;&#xB824;&#xBA74; &#xB450; &#xBC88;&#xC9F8; &#xBC0F; &#xC138; &#xBC88;&#xC9F8; &#xC5FC;&#xC0C9;&#xCCB4;&#xC5D0; 124 &#xACB0;&#xD568; &#xBC0F; &#xB450; &#xBC88;&#xC9F8; &#xC5FC;&#xC0C9;&#xCCB4;&#xC5D0; 14 &#xC911;&#xBCF5; &#xAC00;&#xB2A5;&#xD55C; &#xB300;&#xB9BD; &#xC720;&#xC804;&#xC790;, hntpeb;&#xC5D0; &#xC758;&#xD574; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC628;&#xB3C4; &#xBBFC;&#xAC10;&#xD55C; &#xAC70;&#xCE5C; &#xB208; &#xD615;&#xC758; &#xBCF5;&#xC6A9;&#xB7C9;&#xC5D0; &#xBBFC;&#xAC10;&#xD55C; &#xC218;&#xC815;&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC11D; &#xD588;&#xB2E4; 29 &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC601;&#xC5ED; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xB2E4;. &#xADF8; &#xD6C4;, 438 P &#xC694;&#xC18C; &#xC720;&#xB3C4; &#xCE58;&#xBA85;&#xC801;&#xC778; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC774; &#xC9C0;&#xC5ED;&#xACFC; 12 &#xD6C4;&#xBCF4; &#xC720;&#xC804;&#xC790;&#xB97C; &#xB9E4;&#xD551; 63 &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xD55C;&#xC815;&#xC790; loci&#xC758; &#xC2DD;&#xBCC4;&#xC73C;&#xB85C; &#xC774;&#xC5B4;&#xC9C0;&#xB294; &#xC720;&#xC804;&#xC790; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0; &#xB300; &#xD55C; &#xD14C;&#xC2A4;&#xD2B8; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBC1C;&#xACAC;&#xB41C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xD558;&#xC704; &#xC9D1;&#xD569; &#xB610;&#xD55C; &#xC9C0;&#xBC30;&#xC801; hnt308&#xC744; &#xC774;&#xC6A9;&#xD55C; &#xBC30;&#xC544; &#xCE58; &#xC0AC; &#xC728;&#xC744; &#xC218;&#xC815; &#xD558; &#xACE0; &#xB530;&#xB77C;&#xC11C; &#xC77C;&#xBC18; &#xBCF4;&#xB2E4;&#xB294; &#xC870;&#xC9C1; &#xAD00;&#xB828; &#xC778;&#xD130; &#xB300;&#xD45C;. &#xC77C;&#xBC18; &#xC778;&#xD130; loci &#xC778;&#xCF54;&#xB529; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC694;&#xC778;, &#xB9D0;&#xB77C; &#xBC14;&#xC778;&#xB529; &#xB2E8;&#xBC31;&#xC9C8;, &#xC2E0;&#xD638; &#xC804;&#xB2EC; &#xB2E8;&#xBC31;&#xC9C8; &#xBC0F; &#xC138;&#xD3EC; &#xC678; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4;&#xC758; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB97C; &#xD3EC;&#xD568; &#xD569;&#xB2C8;&#xB2E4;. &#xD45C;&#xD604;&#xC758; &#xC5EC;&#xB7EC; &#xC778;&#xD130; hnt &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xD3C9;&#xAC00; &#xB418;&#xC5C8;&#xB2E4;. 5 &#xAC1C;&#xC758; &#xC720;&#xC804;&#xC790;-apontic (apt), &#xB378;&#xD0C0; (Dl), decapentaplegic (dpp), karst (kst)&#xC640; &#xD55C; (puc)-&#xC790;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xADDC;&#xC81C; &#xC870;&#xC9C1; &#xB418; &#xACE0; &#xB530;&#xB77C;&#xC11C;, HNT&#xC758; &#xC9C1;&#xC811; transcriptional &#xB300;&#xC0C1;&#xC774; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. 3&#xC774; &#xC720;&#xC804;&#xC790;-apt, Dl, &#xBC0F; dpp-&#xB610;&#xD55C; &#xC778;&#xC811; HNT &#xD45C;&#xD604; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; nonautonomously &#xADDC;&#xC81C; &#xB429;&#xB2C8;&#xB2E4;. &#xBA87; &#xAC00;&#xC9C0; &#xCD94;&#xAC00; &#xC778;&#xD130;-&#xBC14;&#xC774;&#xD0B9; (vkg), Cg25, &#xBC0F; laminin &#xC54C;&#xD30C; (&#xB77C; &#xB098;) &#xC2DD;-nonautonomous &#xBC29;&#xC2DD;&#xC5D0;&#xB9CC; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xBCF5;&#xC6A9;&#xB7C9;&#xC5D0; &#xBBFC;&#xAC10;&#xD55C; autosomal &#xD55C;&#xC815;&#xC790; &#xCD08;&#xD30C;&#xB9AC; &#xD575; &#xC544;&#xC5F0; &#xD551;&#xAC70; &#xB2E8;&#xBC31;&#xC9C8;, &#xB4B7; &#xAD81;&#xB9AC;&#xC5D0; &#xC758;&#xD574; &#xC790;&#xCE58; &#xC870;&#xC9C1;&#xACFC; &#xC870;&#xC9C1; nonautonomous &#xB808; &#xADE4; &#xB808;&#xC774; &#xC158;&#xC5D0; &#xB300; &#xD55C; &#xD6C4;&#xBCF4; &#xC720;&#xC804;&#xC790;&#xB97C; &#xC2DD;&#xBCC4;&#xD569;&#xB2C8;&#xB2E4;.

La prote&iacute;na nuclear zinc-finger, codificada por el locus retrospectiva (hnt) regula varios procesos celulares en el epitelio de la Drosophila, incluyendo la quinasa Jun N-terminal (JNK) se&ntilde;alizaci&oacute;n v&iacute;a y actinia polimerizaci&oacute;n. Defectos en estas v&iacute;as moleculares pueden subyacer las interacciones celulares anormales, p&eacute;rdida de la integridad epitelial y apoptosis que ocurre en hnt mutantes, que a su vez causa falta de procesos morfogen&eacute;ticos como germen banda retracci&oacute;n y cierre dorsal en el embri&oacute;n. Para definir las v&iacute;as gen&eacute;ticas reguladas por hnt, 124 deficiencias en la segunda y terceros cromosomas y 14 duplicaciones en el segundo cromosoma fueron probadas para la modificaci&oacute;n de la dosis-sensible de un fenotipo termosensible &aacute;spero ojo causado por el alelo viable, hntpeb; se identificaron 29 regiones de interacci&oacute;n. Posteriormente, 438 P-elemento-inducido mutaciones letales asignar a estas regiones y genes candidatos 12 fueron probadas para la interacci&oacute;n gen&eacute;tica, llevando a la identificaci&oacute;n de 63 loci modificador dominante. Un subconjunto de los mutantes identificados tambi&eacute;n dominante modificar letalidad embrionaria inducida por hnt308 y as&iacute; representan general en lugar de interactianos tejidos espec&iacute;ficos. Interactianos generales incluyen lugares geom&eacute;tricos que codifican factores de transcripci&oacute;n, prote&iacute;nas de uni&oacute;n a actina, prote&iacute;nas de transducci&oacute;n de se&ntilde;al y componentes de la matriz extracelular. Se evalu&oacute; la expresi&oacute;n de interactianos varios en tejido mutante hnt. Cinco genes--apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst) y arrugado o fruncido (puc)--son tejido regulado aut&oacute;nomo y, as&iacute;, puede ser directas objetivos transcripcionales de HNT. Tres de estos genes--aptos, Dl y dpp--est&aacute;n regulados tambi&eacute;n nonautonomously en los tejidos adyacentes no-HNT-expresi&oacute;n. La expresi&oacute;n de varios interactianos adicionales--viking (vkg), Cg25 y laminin-alfa (LanA)-depende s&oacute;lo de una manera no aut&oacute;nomos.

Sensible a la dosis un autosoma modificadores identifican genes candidatos para tejido aut&oacute;nomo y no aut&oacute;nomos Reglamento de tejido por la prote&iacute;na nuclear zinc-finger de Drosophila, retrospectiva.

Aven is a regulator of the DNA-damage response and G2/M cell cycle progression. Overexpression of Aven is associated with poor prognosis in patients with childhood acute lymphoblastic leukemia and acute myeloid leukemia, and altered intracellular Aven distribution is associated with inﬁltrating ductal carcinoma and papillary carcinoma breast cancer subtypes. Although Aven orthologs have been identified in most vertebrate species, no Aven gene has been reported in invertebrates. Here, we describe a Drosophila melanogaster open reading frame (ORF) that shares sequence and functional similarities with vertebrate Aven genes. The protein encoded by this ORF, which we named dAven, contains several domains that are highly conserved among Aven proteins of fish, amphibian, bird and mammalian origins. In flies, knockdown of dAven by RNA interference (RNAi) resulted in lethality when its expression was reduced either ubiquitously or in fat cells using Gal4 drivers. Animals undergoing moderate dAven knockdown in the fat body had smaller fat cells displaying condensed chromosomes and increased levels of the mitotic marker phosphorylated histone H3 (PHH3), suggesting that dAven was required for normal cell cycle progression in this tissue. Remarkably, expression of dAven in Xenopus egg extracts resulted in G2/M arrest that was comparable to that caused by human Aven. Taken together, these results suggest that, like its vertebrate counterparts, dAven plays a role in cell cycle regulation. Drosophila could be an excellent model for studying the function of Aven and identifying cellular factors that influence its activity, revealing information that may be relevant to human disease.

Identification of dAven, a Drosophila melanogaster ortholog of the cell cycle regulator Aven.

The transmembrane proteoglycan Syndecan contributes to cell surface signaling of diverse ligands in mammals, yet in Drosophila, genetic evidence links Syndecan only to the Slit receptor Roundabout and to the receptor tyrosine phosphatase LAR. Here we characterize the requirement for syndecan in the determination and morphogenesis of the Drosophila heart, and reveal two phases of activity, indicating that Syndecan is a co-factor in at least two signaling events in this tissue. There is a stochastic failure to determine heart cell progenitors in a subset of abdominal hemisegments in embryos mutant for syndecan, and subsequent to Syndecan depletion by RNA interference. This phenotype is sensitive to gene dosage in the FGF receptor (Heartless), its ligand, Pyramus, as well as BMP-ligand Decapentaplegic (Dpp) and co-factor Sara. Syndecan is also required for lumen formation during assembly of the heart vessel, a phenotype shared with mutations in the Slit and Integrin signaling pathways. Phenotypic interactions of syndecan with slit and Integrin mutants suggest intersecting function, consistent with Syndecan acting as a co-receptor for Slit in the Drosophila heart.

Syndecan contributes to heart cell specification and lumen formation during Drosophila cardiogenesis.

Previous work has shown that many of the 18 family members of Drosophila nuclear receptor transcription factors function in a temporal hierarchy to coordinate developmental progression and growth with the rate limiting process of metabolism. To gain further insight into these interactions and processes, we have undertaken a whole-family analysis of nuclear receptor mRNA spatial expression patterns over the entire process of embryogenesis, as well as the 3rd instar wandering larva stage, by using high-resolution fluorescence in situ hybridization. Overall, the patterns of expression are remarkably consistent with previously mapped spatial activity profiles documented during the same time points, with similar hot spots and temporal profiles in endocrine and metabolically important tissues. Among the more remarkable of the findings is that the majority of mRNA expression patterns observed show striking subcellular distributions, indicating potentially critical roles in the control of protein synthesis and subsequent subcellular distributions. These patterns will serve as a useful reference for future studies on the tissue-specific roles and interactions of nuclear receptor proteins, partners, cofactors and ligands.

Spatial profiling of nuclear receptor transcription patterns over the course of Drosophila development.

The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. Here we investigate HNT's functional motifs, target genes and its regulatory abilities. We show that the C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. We map HNT's in vivo binding sites on salivary gland polytene chromosomes and define, at high resolution, where HNT is bound to two target genes, hnt itself and nervy (nvy). Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNT's ninth Zinc finger has degenerated or been lost in the vertebrate lineage. We show that a HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control.

Drosophila Hindsight and mammalian RREB-1 are evolutionarily conserved DNA-binding transcriptional attenuators.

Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1-actomyosin activity. Recently, we found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. Here, we report that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo . These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses we found that Step normally activates Arf small G proteins and antagonizes Rho1-actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, we examined the effect of Step on PGC formation at the posterior pole. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP-Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. We propose that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.

Germ Cell Segregation from the Drosophila Soma Is Controlled by an Inhibitory Threshold Set by the Arf-GEF Steppke.

Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. In the present study, we have examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, we generated two independent excisions of nemy that differentially affect the expression of nemy isoforms. We show that the nemy45 excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. We further show that nemy45 mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine β hydroxylase activity (TβH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators.

Distinct Regulation of Transmitter Release at the Drosophila NMJ by Different Isoforms of nemy.

In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided.

Ronit Wilk

The Terrence Donnelly Centre for Cellular and Biomolecular Research,
Department of Molecular Genetics

University of Toronto

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Ronit Wilk

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Terrence Donnelly Centre for Cellular and Biomolecular Research,Department of Molecular Genetics

University of Toronto

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The Terrence Donnelly Centre for Cellular and Biomolecular Research,
Department of Molecular Genetics