Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

University of North Carolina, Chapel Hill

The development of the Drosophila visual system utilizes two members of the highly conserved Six-Homeobox family of transcription factor, Sine oculis and Optix. Although in vitro studies have detected differences in DNA-binding and interactions with some co-factors, questions remain as to what extent the activity for these two transcriptional regulators is redundant or specific in vivo. In this work, we show that the SoD mutation within the Six domain does not abolish DNA-protein interactions, but alters co-factor binding specificity to resemble that of Optix. A mutation in the same region of Optix alters its activity in vivo. We propose that the dominant mutant phenotype is primarily due to an alteration in binding properties of the Sine oculis protein and that distinct partner interactions is one important mechanism in determining significant functional differences between these highly conserved factors during eye development.

Partner specificity is essential for proper function of the SIX-type homeodomain proteins Sine oculis and Optix during fly eye development.

Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins, CRX, OTX1, and OTX2, fulfill these functions. In Drosophila, the single founding member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. We have used global gene expression analysis in late pupal heads to better characterize the post-embryonic functions of Otd in Drosophila. We have identified 61 genes that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one-third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well-documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes, notwithstanding the significant divergence of the underlying genetic pathways. Our findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria.

Analysis of the Otd-dependent transcriptome supports the evolutionary conservation of CRX/OTX/OTD functions in flies and vertebrates.

&#x73B0;&#x6709;&#x7684;&#x57FA;&#x4E8E;&#x957F;&#x53CC;&#x7EDE;&#x53D1;&#x5939;&#x679C;&#x8747;&#x7684;&#x8F6C;&#x57FA;&#x56E0; RNAi &#x8D44;&#x6E90; RNAs &#x529F;&#x80FD;&#x7814;&#x7A76;&#x7684;&#x6709;&#x529B;&#x5DE5;&#x5177;&#xFF0C;&#x4F46;&#x90FD;&#x65E0;&#x6548;&#x7684;&#x57FA;&#x56E0;&#x6572;&#x5728;&#x5375;&#x5B50;&#x53D1;&#x751F;&#xFF0C;&#x8BB8;&#x591A;&#x751F;&#x7269;&#x95EE;&#x9898;&#x7814;&#x7A76;&#x7684;&#x91CD;&#x8981;&#x6A21;&#x578B;&#x7CFB;&#x7EDF;&#x3002;&#x6211;&#x4EEC;&#x8868;&#x660E; shRNAs&#xFF0C;&#x5185;&#x6E90;&#x6027; microRNA &#x4E3A;&#x84DD;&#x672C;&#x662F;&#x6781;&#x4E3A;&#x6709;&#x6548;&#x7684;&#x6C89;&#x9ED8;&#x5375;&#x5B50;&#x53D1;&#x751F;&#x8FC7;&#x7A0B;&#x4E2D;&#x7684;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x4ECB;&#x7ECD;&#x4E86;&#x6211;&#x4EEC;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x7684; shRNA &#x8D44;&#x6E90;&#x5EFA;&#x8BBE;&#x7684;&#x8FDB;&#x5EA6;&#x3002;

&#x8F6C;&#x57FA;&#x56E0; RNAi &#x5728;&#x679C;&#x8747;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x89C4;&#x6A21; shRNA &#x8D44;&#x6E90;&#x3002;

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

A genome-scale shRNA resource for transgenic RNAi in Drosophila.

Les ressources de RNAi transg&eacute;niques chez Drosophila melanogaster, bas&eacute; sur l&#39;&eacute;pingle &agrave; cheveux long double-brin RNAs sont des outils puissants pour des &eacute;tudes fonctionnelles, mais ils sont inefficaces dans de g&egrave;ne au cours de l&#39;ovogen&egrave;se, un important syst&egrave;me mod&egrave;le pour l&#39;&eacute;tude de nombreuses questions biologiques. Nous montrons que les interf&eacute;rents, calqu&eacute;s sur un micro-ARN endog&egrave;ne, sont extr&ecirc;mement efficaces &agrave; r&eacute;duire au silence l&#39;expression des g&egrave;nes au cours de l&#39;ovogen&egrave;se. Nous d&eacute;crivons &eacute;galement nos progr&egrave;s &agrave; la cr&eacute;ation d&#39;une ressource de shARN genome-wide.

Une ressource de shARN &eacute;chelle g&eacute;nomique pour RNAi transg&eacute;nique chez la drosophile.

Vorhandene transgene RNAi-Ressourcen in Drosophila Melanogaster basierend auf lange Doppelstrang-Haarnadel RNAs sind leistungsstarke Tools f&uuml;r Funktionsstudien, aber sie sind ineffektiv in gen Niederschlag w&auml;hrend der Oogenese, ein wichtiges Modell-System f&uuml;r die Studie der vielen biologischen Fragen. Wir zeigen, dass ShRNAs, modelliert auf eine endogene MicroRNA, &auml;u&szlig;erst wirksam bei Unterdr&uuml;ckender Genexpression w&auml;hrend der Oogenese. Wir beschreiben auch unser Fortschritt beim Aufbau einer genomweiten ShRNA-Ressource.

Eine Genom-Skala-ShRNA-Ressource f&uuml;r transgene RNAi in Drosophila.

Esistenti risorse RNAi transgeniche in Drosophila melanogaster basato sul tornante lungo double-stranded RNAs sono potenti strumenti per studi funzionali, ma sono inefficaci del gene durante l&#39;ovogenesi, un importante modello di sistema per lo studio di molte questioni biologiche. Mostriamo che shRNAs, modellato su un microRNA endogeno, sono estremamente efficaci a tacere espressione genica durante l&#39;ovogenesi. Descriviamo anche il nostro progresso verso la creazione di una risorsa di shRNA genome-wide.

Una risorsa di shRNA genoma-scala per RNAi transgenici in drosofila.

&#x65E2;&#x5B58;&#x306E;&#x9577;&#x3044;&#x4E8C;&#x672C;&#x9396;&#x30D8;&#x30A2;&#x30D4;&#x30F3;&#x578B;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x30AD;&#x30A4;&#x30ED;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306B;&#x304A;&#x3051;&#x308B;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; RNAi &#x30EA;&#x30BD;&#x30FC;&#x30B9; Rna &#x6A5F;&#x80FD;&#x7814;&#x7A76;&#x306E;&#x5F37;&#x529B;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3067;&#x3059;&#x304C;&#x5F7C;&#x3089;&#x306F;&#x5375;&#x3001;&#x591A;&#x304F;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x8CEA;&#x554F;&#x306E;&#x7814;&#x7A76;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30E2;&#x30C7;&#x30EB; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x907A;&#x4F1D;&#x5B50;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3;&#x3067;&#x52B9;&#x679C;&#x7684;&#x306A;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;&#x6211;&#x3005; &#x306F;&#x5185;&#x56E0;&#x6027;&#x30DE;&#x30A4;&#x30AF;&#x30ED; Rna &#x3092;&#x30E2;&#x30C7;&#x30EB;&#x5316;&#x3057;&#x305F; Shrna &#x304C;&#x5375;&#x5F62;&#x6210;&#x4E2D;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3092;&#x9ED9;&#x3089;&#x305B;&#x308B;&#x3053;&#x3068;&#x306B;&#x6975;&#x3081;&#x3066;&#x52B9;&#x679C;&#x7684;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x30B2;&#x30CE;&#x30E0; shRNA &#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x306E;&#x69CB;&#x7BC9;&#x306B;&#x5411;&#x3051;&#x3066;&#x6211;&#x3005; &#x306E;&#x9032;&#x6B69;&#x306B;&#x3064;&#x3044;&#x3066;&#x3082;&#x8FF0;&#x3079;&#x308B;&#x3002;

&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; RNAi &#x306E;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x898F;&#x6A21;&#x306E; shRNA &#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3002;

&#xAE30;&#xC874; Drosophila melanogaster &#xAE34; &#xB354;&#xBE14;-&#xC88C;&#xCD08; &#xBA38;&#xB9AC; &#xD540; &#xAE30;&#xBC18;&#xC5D0;&#xC11C; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; RNAi &#xB9AC;&#xC18C;&#xC2A4; RNAs &#xAE30;&#xB2A5; &#xC5F0;&#xAD6C;&#xB97C; &#xC704;&#xD55C; &#xAC15;&#xB825;&#xD55C; &#xB3C4;&#xAD6C; &#xD558;&#xC9C0;&#xB9CC; &#xADF8;&#xB4E4;&#xC740; &#xC720;&#xC804;&#xC790; &#xCD5C;&#xC800;&#xC758;&#xC5D0; &#xD6A8;&#xACFC;&#xC801;&#xC778; oogenesis, &#xB9CE;&#xC740; &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xC9C8;&#xBB38; &#xC5F0;&#xAD6C;&#xB97C; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xBAA8;&#xB378; &#xC2DC;&#xC2A4;&#xD15C; &#xC911;. &#xC6B0;&#xB9AC; &#xC0DD; Microrna&#xB97C; &#xBAA8;&#xB378;&#xB85C; shRNAs oogenesis &#xC911; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC744; &#xCE68;&#xBB35;&#xC5D0; &#xB9E4;&#xC6B0; &#xD6A8;&#xACFC;&#xC801;&#xC778; &#xBCF4;&#xC5EC;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xAC8C;&#xB188; &#xB113;&#xC740; shRNA &#xC790;&#xC6D0; &#xAC74;&#xBB3C;&#xC744; &#xD5A5;&#xD574; &#xC6B0;&#xB9AC;&#xC758; &#xC9C4;&#xD589; &#xC0C1;&#xD669;&#xC744; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;.

&#xCD08;&#xD30C;&#xB9AC; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; Rnai&#xC758; &#xAC8C;&#xB188; &#xADDC;&#xBAA8; shRNA &#xC790;&#xC6D0;.

Existen transg&eacute;nicos recursos de RNAi en Drosophila melanogaster basado en horquilla de largo double-stranded RNAs son poderosas herramientas para estudios funcionales, pero son ineficaces en la ca&iacute;da de gene durante la oog&eacute;nesis, un sistema importante modelo para el estudio de muchas cuestiones biol&oacute;gicas. Mostramos que shRNAs, inspirados en un microARN end&oacute;gena, son muy eficaces en el silenciamiento de la expresi&oacute;n g&eacute;nica durante la oog&eacute;nesis. Tambi&eacute;n describimos nuestro progreso hacia la construcci&oacute;n de un recurso de shRNA genoma.

Un recurso de shRNA genoma-escala para transg&eacute;nico RNAi en Drosophila.

Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.

A regulatory network of Drosophila germline stem cell self-renewal.

To facilitate large scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently comprised of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated web site, the RNAi Stock Validation and Phenotypes Project (RSVP; www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (USA), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

Yang Zhou

Department of Pathology and Laboratory Medicine,
McAllister Heart Institute,
Department of Pathology and Laboratory Medicine, McAllister Heart Institute

University of North Carolina, Chapel Hill

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Yang Zhou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology and Laboratory Medicine,McAllister Heart Institute,Department of Pathology and Laboratory Medicine, McAllister Heart Institute

University of North Carolina, Chapel Hill

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Department of Pathology and Laboratory Medicine,
McAllister Heart Institute,
Department of Pathology and Laboratory Medicine, McAllister Heart Institute