setting-up-simple-light-sheet-microscope-for-toto-imaging-c-elegans

Setting Up a Simple Light Sheet Microscope for <em>In Toto</em> Imaging of <em>C. elegans</em> Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

UMR7288 CNRS, Aix-Marseille Université

In chordates, formation of neural tissue from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in vertebrates. Here, using the early neural marker Otx as an entry point, we dissected the neural induction pathway in the simple embryos of Ciona intestinalis. We first isolated the regulatory element driving Otx expression in the prospective neural tissue, showed that this element directly responds to FGF signaling and that FGF9/16/20 acts as an endogenous neural inducer. Binding site analysis and gene loss of function established that FGF9/16/20 induces neural tissue in the ectoderm via a synergy between two maternal response factors. Ets1/2 mediates general FGF responsiveness, while the restricted activity of GATAa targets the neural program to the ectoderm. Thus, our study identifies an endogenous FGF neural inducer and its early downstream gene cascade. It also reveals a role for GATA factors in FGF signaling.

Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors.

Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis, which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, we compared in both species the regulation of Otx, a gene with a shared complex expression pattern. We found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved, in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. We propose that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates.

Making very similar embryos with divergent genomes: conservation of regulatory mechanisms of Otx between the ascidians Halocynthia roretzi and Ciona intestinalis.

The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates.

A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

Our understanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Our cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal beta-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ci-fog is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. We thus identify a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program.

A combinatorial code of maternal GATA, Ets and beta-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm.

How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. We establish here a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/beta-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. Our study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/beta-catenin pathway is widely used to control the identity of neuronal lineages.

Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans.

Wnt asymmetry and the terminal division of neuronal progenitors.

&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x6D4B;&#x5E8F; (WGS) &#x663E;&#x793A;&#x7279;&#x5B9A;&#x7684;&#x7A81;&#x53D8;&#x578B;&#x751F;&#x7269;&#x7684;&#x662F;&#x5F3A;&#x5927;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x627E;&#x51FA;&#x8FC7;&#x591A;&#x7684;&#x751F;&#x7269;&#x8FC7;&#x7A0B;&#x7684;&#x9057;&#x4F20;&#x51B3;&#x5B9A;&#x56E0;&#x7D20;&#x3002;&#x6211;&#x4EEC;&#x4EE5;&#x524D;&#x901A;&#x8FC7;&#x786E;&#x5B9A;&#x8D1F;&#x8D23;&#x4E00;&#x4E2A;&#x7279;&#x5B9A;&#x7684;&#x8868;&#x578B;&#xFF0C;&#x4E59;&#x57FA;&#x7532;&#x57FA;&#x78FA;&#x9178; (EMS) &#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x7A81;&#x53D8;&#x70B9;&#x8F68;&#x8FF9;&#x9A8C;&#x8BC1;&#x8FD9;&#x79CD;&#x65B9;&#x6CD5;&#x7684;&#x53EF;&#x884C;&#x6027;-&#x8BF1;&#x53D8;&#x7C73;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x5E94;&#x53D8;&#x3002;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x4ECB;&#x7ECD; 17 EMS &#x8BF1;&#x53D8;&#x7C73;&#x57FA;&#x56E0;&#x7EC4;&#x7684;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x7A81;&#x53D8;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#xFF0C;&#x6240;&#x8BC4;&#x4F30;&#x4E0E;&#x751F;&#x7269;&#x4FE1;&#x606F;&#x5B66;&#x7684;&#x7BA1;&#x9053;&#xFF0C;&#x79F0;&#x4E3A; MAQGene&#x3002;&#x51FA;&#x4EBA;&#x610F;&#x6599;&#x7684;&#x662F;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#xFF0C;&#x867D;&#x7136;&#x5F02;&#x4EA4;&#x7ED3;&#x5B9E; mutagenized &#x7684;&#x682A;&#x4E0D;&#x4F1A;&#x51CF;&#x5C11;&#x7A81;&#x53D8;&#x7684;&#x603B;&#x6570;&#xFF0C;&#x60CA;&#x4EBA;&#x7684;&#x7A81;&#x53D8;&#x8D1F;&#x8F7D;&#x662F;&#x4ECD;&#x6307;&#x51FA;&#x5373;&#x4F7F;&#x5728; outcrossed &#x682A;&#x3002;&#x8FD9;&#x79CD;&#x9057;&#x4F20;&#x7684;&#x590D;&#x6742;&#x6027;&#x6709;&#x5EFA;&#x7ACB;&#x57FA;&#x56E0;&#x578B;&#x4E0E;&#x8868;&#x578B;&#x4E4B;&#x95F4;&#x7684;&#x56E0;&#x679C;&#x5173;&#x7CFB;&#x65F6;&#x5FC5;&#x987B;&#x8003;&#x8651;&#x5230;&#x3002;&#x5373;&#x4F7F;&#x65E0;&#x610F;&#xFF0C;&#x6B64;&#x5904;&#x6240;&#x8FF0;&#x7684; 17 &#x987A;&#x5E8F;&#x7684;&#x682A;&#x63D0;&#x4F9B;&#x51E0;&#x4E4E; 1000&#x5E74;&#x57FA;&#x56E0;&#xFF0C;&#x5305;&#x62EC; 62 &#x63D0;&#x524D;&#x7EC8;&#x6B62;&#x5BC6;&#x7801;&#x5B50;&#xFF0C;&#x4EE3;&#x8868;&#x5019;&#x9009;&#x4EBA;&#x6316;&#x7A7A;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#xFF0C;&#x5C06;&#x8FDB;&#x4E00;&#x6B65;&#x4E3A;&#x7EBF;&#x866B;&#x793E;&#x4F1A;&#x7814;&#x7A76;&#x57FA;&#x56E0;&#x529F;&#x80FD;&#x7684;&#x4F7F;&#x7528;&#x7684;&#x57FA;&#x56E0;&#x591A;&#x6001;&#x6027;&#x7684;&#x8D44;&#x6E90;&#x3002;

&#x591A;&#x4E2A;&#x4E59;&#x57FA;&#x7532;&#x57FA;&#x78FA;&#x9178;&#x8BF1;&#x53D8;&#x7C73;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x682A;&#x7684;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x6D4B;&#x5E8F;&#x5206;&#x6790;&#x3002;

Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.

Analysis of multiple ethyl methanesulfonate-mutagenized Caenorhabditis elegans strains by whole-genome sequencing.

Le s&eacute;quen&ccedil;age du g&eacute;nome entier (WGS) d&#39;organismes affichant un ph&eacute;notype mutant sp&eacute;cifique est une approche puissante pour identifier les d&eacute;terminants g&eacute;n&eacute;tiques d&#39;une multitude de processus biologiques. Nous avons d&eacute;j&agrave; valid&eacute; la faisabilit&eacute; de cette approche en identifiant un locus point-une mutation responsable d&#39;un ph&eacute;notype sp&eacute;cifique, observ&eacute; dans un m&eacute;thanesulfonate d&#39;&eacute;thyle (EMS)-mutag&eacute;nis&eacute;es tant Caenorhabditis elegans souche. Nous d&eacute;crivons ici le profil mutationnel G&eacute;nome-large de 17 g&eacute;nomes EMS-mutag&eacute;nis&eacute;es tant tel qu&#39;&eacute;valu&eacute; par un pipeline de bioinformatique, appel&eacute; MAQGene. &Eacute;tonnamment, nous constatons que, tandis que le croisement de souches mutag&eacute;nis&eacute;s r&eacute;duit le nombre de mutations, une charge mutationnelle frappante est encore observ&eacute;e m&ecirc;me dans des souches. Une telle complexit&eacute; g&eacute;n&eacute;tique doit &ecirc;tre pris en compte lors de l&#39;&eacute;tablissement d&#39;un rapport causal entre le g&eacute;notype et le ph&eacute;notype. Bien qu&#39;involontaire, s&eacute;quenc&eacute;es 17 souches d&eacute;crites ici constituent une ressource de variants all&eacute;liques dans pr&egrave;s de 1000 g&egrave;nes, dont 62 codons stop pr&eacute;matur&eacute;s, qui repr&eacute;sentent des all&egrave;les de knock-out candidat qui seront plus utile pour la communaut&eacute; de c. elegans afin d&#39;&eacute;tudier la fonction des g&egrave;nes.

Analyse de l&#39;&eacute;thyl-mutag&eacute;nis&eacute;es tant m&eacute;thanesulfonate Caenorhabditis elegans souches multiples par s&eacute;quen&ccedil;age de g&eacute;nomes complets.

Ganz-Genoms (WGS) Organismen, die Darstellung eines bestimmten Mutanten Ph&auml;notyps ist einen leistungsf&auml;higen Ansatz um die genetischen Determinanten der eine Vielzahl von biologischen Prozessen zu identifizieren. Wir haben zuvor validiert die Machbarkeit dieses Ansatzes durch die Identifizierung einer Punkt-mutiert Locus verantwortlich f&uuml;r einen bestimmten Ph&auml;notyp, beobachtet in einem Ethylmethansulfonat (EMS)-mutagenized Caenorhabditis Elegans Belastung. Hier beschreiben wir das genomweiten teilhabende Profil 17 EMS-mutagenized Genome mit einer Bioinformatic Pipeline, genannt MAQGene zu bewerten. &Uuml;berraschenderweise wird feststellen, dass wir zwar Auskreuzung mutagenized St&auml;mme die Gesamtzahl der Mutationen, eine markante teilhabende Last noch selbst bei den outcrossed St&auml;mmen beobachtet. Solche genetische Komplexit&auml;t muss ber&uuml;cksichtigt werden, wenn eine urs&auml;chliche Beziehung zwischen Genotyp und Ph&auml;notyp zur Gr&uuml;ndung. Obwohl unbeabsichtigt, die 17 sequenzierte St&auml;mme, die hier beschriebenen bieten eine Ressource von allelische Varianten in fast 1000 Genen, darunter 62 vorzeitiges Stop-Codons, die Kandidaten k.o. Allele darstellen, die von weiteren Nutzen f&uuml;r die Gemeinschaft von C. Elegans Genfunktion zu studieren.

Analyse von mehreren Ethyl Methansulfonat-mutagenized Caenorhabditis Elegans St&auml;mmen durch Sequenzierung des gesamten Genoms.

Whole-genome sequencing (WGS) di visualizzazione di un fenotipo mutante specifico organismi &egrave; un potente approccio per identificare i determinanti genetici di una pletora di processi biologici. Noi abbiamo gi&agrave; convalidati la fattibilit&agrave; di questo approccio identificando un locus mutato punto responsabile un fenotipo specifico, osservato in un etile Metansulfonato (EMS)-mutagenized Caenorhabditis elegans ceppo. Qui descriviamo il profilo mutazionale genome-wide di genomi EMS-mutagenized 17 come valutati con una pipeline di bioinformatica, chiamata MAQGene. Sorprendentemente, troviamo che mentre outcrossing ceppi mutagenized ridurre il numero totale delle mutazioni, un notevole carico mutazionale &egrave; ancora osservato anche in ceppi outcrossed. Tale complessit&agrave; genetica deve essere preso in considerazione quando si stabilisce una relazione causa tra genotipo e fenotipo. Anche se non intenzionale, 17 ceppi sequenziati descritti qui forniscono una risorsa di varianti alleliche in quasi 1000 geni, compreso 62 codoni di stop prematuro, che rappresentano gli alleli di knockout candidato che saranno di ulteriore utilizzo per la comunit&agrave; di c. elegans studiare la funzione del gene.

Analisi delle molteplici etile Metansulfonato mutagenized Caenorhabditis elegans ceppi dal sequenziamento del genoma intero.

&#x5168;&#x30B2;&#x30CE;&#x30E0; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9; (WG) &#x7279;&#x5B9A;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x5F62;&#x8CEA;&#x3092;&#x8868;&#x793A;&#x3059;&#x308B;&#x751F;&#x7269;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x8304;&#x591A;&#x306E;&#x907A;&#x4F1D;&#x306E;&#x6C7A;&#x5B9A;&#x8981;&#x56E0;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x5F37;&#x529B;&#x306A;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3067;&#x3059;&#x3002;&#x6211;&#x3005; &#x4EE5;&#x524D;&#x306E;&#x70B9;&#x5909;&#x7570;&#x8ECC;&#x8DE1;&#x3001;&#x30A8;&#x30C1;&#x30EB; &#x30E1;&#x30BF;&#x30F3;&#x30B9;&#x30EB;&#x30DB;&#x30F3;&#x9178; (EMS) &#x3067;&#x89B3;&#x6E2C;&#x3055;&#x308C;&#x305F;&#x7279;&#x5B9A;&#x306E;&#x8868;&#x73FE;&#x578B;&#x3001;&#x8CAC;&#x4EFB;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x3053;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x691C;&#x8A3C;&#x3057;&#x3066;-&#x7DDA;&#x866B;&#x7DDA;&#x866B;&#x3072;&#x305A;&#x307F;&#x3092;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067; MAQGene &#x3068;&#x547C;&#x3070;&#x308C;&#x308B; bioinformatic &#x30D1;&#x30A4;&#x30D7;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x8A55;&#x4FA1;&#x3057;&#x305F; 17 &#x306E; EMS &#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x5909;&#x7570;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x9A5A;&#x3044;&#x305F;&#x3053;&#x3068;&#x306B;&#x3001;&#x5370;&#x8C61;&#x7684;&#x306A;&#x5909;&#x7570;&#x8CA0;&#x8377;&#x306F;&#x4EBA;&#x70BA;&#x7CFB;&#x7D71;&#x30A2;&#x30A6;&#x30C8;&#x30AF;&#x30ED;&#x30B9;&#x5909;&#x7570;&#x306E;&#x5408;&#x8A08;&#x6570;&#x3092;&#x6E1B;&#x3089;&#x3059;&#x304B;&#x304C;&#x6211;&#x3005; &#x3092;&#x898B;&#x3064;&#x3051;&#x308B;&#x3001;&#x307E;&#x3060;&#x3067;&#x3055;&#x3048; outcrossed &#x83CC;&#x682A;&#x3092;&#x89B3;&#x5BDF;&#x3067;&#x3059;&#x3002;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x907A;&#x4F1D;&#x7684;&#x8907;&#x96D1;&#x3055;&#x306B;&#x907A;&#x4F1D;&#x5B50;&#x578B;&#x304A;&#x3088;&#x3073;&#x8868;&#x73FE;&#x578B;&#x9593;&#x306E;&#x539F;&#x56E0;&#x95A2;&#x4FC2;&#x3092;&#x78BA;&#x7ACB;&#x3059;&#x308B;&#x3068;&#x304D;&#x306B;&#x8003;&#x616E;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x610F;&#x56F3;&#x7684;&#x306B;&#x3053;&#x3053;&#x3067;&#x8AAC;&#x660E;&#x3057;&#x305F; 17 &#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3055;&#x308C;&#x305F;&#x83CC;&#x682A;&#x3067;&#x3055;&#x3089;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x6A5F;&#x80FD;&#x7814;&#x7A76;&#x7DDA;&#x866B;&#x306E;&#x30B3;&#x30DF;&#x30E5;&#x30CB;&#x30C6;&#x30A3;&#x306E;&#x305F;&#x3081;&#x306E;&#x306A;&#x308A;&#x307E;&#x3059;&#x5019;&#x88DC;&#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8;&#x306E;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x8868;&#x3059;&#x3001;62 &#x306E;&#x65E9;&#x671F;&#x505C;&#x6B62;&#x30B3;&#x30C9;&#x30F3;&#x3092;&#x542B;&#x3080;&#x3001;&#x307B;&#x307C; 1000&#x5E74;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x3001;allelic &#x5909;&#x5F62;&#x306E;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x8907;&#x6570;&#x30A8;&#x30C1;&#x30EB; &#x30E1;&#x30BF;&#x30F3;&#x30B9;&#x30EB;&#x30DB;&#x30F3;&#x9178;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x7DDA;&#x866B;&#x7DDA;&#x866B;&#x83CC;&#x682A;&#x306B;&#x3088;&#x3063;&#x3066;&#x5168;&#x30B2;&#x30CE;&#x30E0; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306E;&#x89E3;&#x6790;&#x3002;

&#xC804;&#xCCB4; &#xAC8C;&#xB188; &#xC2DC;&#xD000;&#xC2F1; (WGS) &#xD45C;&#xC2DC; &#xD2B9;&#xC815; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD615; &#xC0DD;&#xBB3C;&#xC758; &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xACFC;&#xC815;&#xC758; &#xACFC;&#xB2E4;&#xC758; &#xC720;&#xC804;&#xC801; &#xACB0;&#xC815; &#xC694;&#xC778; &#xC2DD;&#xBCC4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xAC15;&#xB825;&#xD55C; &#xC811;&#xADFC; &#xC774;&#xB2E4;. &#xC810; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB85C;&#xCEE4; &#xC5D0;&#xD2F8; methanesulfonate (EMS)&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xD2B9;&#xC815; &#xD45C;&#xD604; &#xD615;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784;&#xC744; &#xC2DD;&#xBCC4; &#xD558; &#xC5EC;&#xC774; &#xC811;&#xADFC; &#xBC29;&#xBC95;&#xC758; &#xD0C0;&#xB2F9;&#xC131; &#xAC80;&#xC99D; &#xC774;&#xC804;&#xC5D0; &#xC6B0;&#xB9AC;-mutagenized Caenorhabditis elegans &#xC2A4;&#xD2B8;&#xB808;&#xC778;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xB294; MAQGene &#xB77C;&#xB294; bioinformatic &#xD30C;&#xC774;&#xD504;&#xB77C;&#xC778; &#xD3C9;&#xAC00; 17 EMS mutagenized &#xC720;&#xC804;&#xC790;&#xC758; &#xAC8C;&#xB188; &#xB113;&#xC740; mutational &#xD504;&#xB85C;&#xD544; &#xC124;&#xBA85;. &#xB180;&#xB78D;&#xAC8C;&#xB3C4;, &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xCD1D; &#xC218;&#xB97C; &#xC904;&#xC77C;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4; outcrossing mutagenized &#xAE34;&#xC7A5; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xBC1C;&#xACAC; &#xD558;&#xB294;, &#xB208;&#xC5D0; &#xB744;&#xB294; mutational &#xBD80;&#xD558;&#xB294; &#xC5EC;&#xC804;&#xD788; &#xAD00;&#xCC30; outcrossed &#xBCC0;&#xC885;&#xC5D0;&#xB3C4;. &#xC774;&#xB7EC;&#xD55C; &#xC720;&#xC804;&#xC790; &#xBCF5;&#xC7A1;&#xC131; &#xC720;&#xC804;&#xC790; &#xD615; &#xBC0F; &#xD45C;&#xD604; &#xD615;&#xC758; &#xC6D0;&#xC778; &#xAD00;&#xACC4;&#xB97C; &#xC124;&#xC815;&#xD560; &#xB54C; &#xACE0;&#xB824; &#xB418;&#xC5B4;&#xC57C; &#xD55C;&#xB2E4;. &#xBE44;&#xB85D; &#xC758;&#xB3C4; &#xD558;&#xC9C0; &#xC54A;&#xC740;, &#xC5EC;&#xAE30;&#xC5D0; &#xC124;&#xBA85; &#xB41C; 17 &#xC2DC;&#xD000;&#xC2A4; &#xBCC0;&#xC885; 62 &#xC870; &#xC815;&#xC9C0; codons &#xD6C4;&#xBCF4; &#xB179;&#xC544;&#xC6C3; &#xB300;&#xB9BD; &#xC720;&#xC804;&#xC790; &#xAE30;&#xB2A5; &#xC5F0;&#xAD6C; C. elegans &#xCEE4;&#xBBA4;&#xB2C8;&#xD2F0;&#xC5D0; &#xB300; &#xD55C; &#xB354; &#xC0AC;&#xC6A9; &#xB420; &#xAC83;&#xC785;&#xB2C8;&#xB2E4; &#xADF8;&#xB97C; &#xB300;&#xD45C; &#xD558;&#xB294; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xAC70;&#xC758; 1000 &#xC720;&#xC804;&#xC790; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xB9AC;&#xC18C;&#xC2A4;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

&#xC5EC;&#xB7EC; &#xC5D0;&#xD2F8; methanesulfonate mutagenized Caenorhabditis elegans &#xC885;&#xC790;&#xC758; &#xC804;&#xCCB4; &#xAC8C;&#xB188; &#xC2DC;&#xD000;&#xC2F1; &#xD558; &#xC5EC; &#xBD84;&#xC11D; &#xD569;&#xB2C8;&#xB2E4;.

La secuencia de genoma completo (WGS) de organismos mostrando un fenotipo mutante espec&iacute;fico es un enfoque de gran alcance para identificar los determinantes gen&eacute;ticos de una gran cantidad de procesos biol&oacute;gicos. Previamente hemos validado la viabilidad de este enfoque mediante la identificaci&oacute;n de un locus mutado de punto responsable de un fenotipo espec&iacute;fico, observado en un Metanosulfonato de etilo (EMS)-mutagenized tensi&oacute;n de Caenorhabditis elegans. Aqu&iacute; describimos el perfil mutacional del genoma de los genomas de EMS-mutagenized 17 evaluada con un gasoducto de Bioinform&aacute;tica, llamado MAQGene. Sorprendentemente, encontramos mientras la exogamia cepas mutagenized reducir el n&uacute;mero de mutaciones, una llamativa carga mutacional todav&iacute;a observa incluso en cepas outcrossed. Tal complejidad gen&eacute;tica debe tenerse en cuenta al establecer una relaci&oacute;n causal entre el genotipo y fenotipo. Aunque no intencional, las cepas secuenciadas 17 descritas aqu&iacute; proveen un recurso de variantes al&eacute;licas en casi 1000 genes, incluyendo 62 codones de parada prematuro, que representan los alelos de octavos de final del candidato que ser&aacute;n de m&aacute;s utilidad para la comunidad de C. elegans estudiar la funci&oacute;n del gen.

An&aacute;lisis de m&uacute;ltiples ethyl methanesulfonate-mutagenized Caenorhabditis elegans cepas por secuenciaci&oacute;n del genoma completo.

Lineage-based mechanisms are widely used to generate cell type diversity in both vertebrates and invertebrates. For the past few decades, the nematode Caenorhabditis elegans has served as a primary model system to study this process because of its fixed and well-characterized cell lineage. Recent studies conducted at the level of single cells and individual cis-regulatory elements suggest a general model by which cellular diversity is generated in this organism. During its developmental history a cell passes through multiple transient regulatory states characterized by the expression of specific sets of transcription factors. The transition from one state to another is driven by a general binary decision mechanism acting at each successive division in a reiterative manner and ending up with the activation of the terminal differentiation program upon terminal division. A similar cell fate specification system seems to play a role in generating cellular diversity in the nervous system of more complex organisms such as Drosophila and vertebrates.

Lineage programming: navigating through transient regulatory states via binary decisions.

Although nervous systems are largely bilaterally symmetric on a structural level, they display striking degrees of functional left/right (L/R) asymmetry. In Caenorhabditis elegans, two structurally symmetric pairs of sensory neurons, ASE and AWC, display two distinctly controlled types of functional L/R asymmetries (stereotyped versus stochastic asymmetry). Beyond these two cases, the extent of neuronal asymmetry in the C. elegans nervous system was unclear. Here, we report that the Beta3/Olig-type bHLH transcription factor hlh-16 is L/R asymmetrically expressed in several distinct, otherwise bilaterally symmetric interneuron and motoneuron pairs that are part of a known navigation circuit. We find that hlh-16 asymmetry is generated during gastrulation by an asymmetric LAG-2/Delta signal originating from the mesoderm that promotes hlh-16 expression in neurons on the left side through direct binding of the Notch effector LAG-1/Su(H)/CBF to a cis-regulatory element in the hlh-16 locus. Removal of hlh-16 reveals an unanticipated asymmetry in the ability of the axons of the AIY interneurons to extend into the nerve ring, with the left AIY axon requiring elevated hlh-16 expression for correct extension. Our study suggests that the extent of molecular L/R asymmetry in the C. elegans nervous system is broader than previously anticipated, establishes a novel signaling mechanism that crosses germ layers to diversify bilaterally symmetric neuronal lineages, and reveals L/R asymmetric control of axonal outgrowth of bilaterally symmetric neurons.

Vincent Bertrand

Institut de Biologie du Développement de Marseille

UMR7288 CNRS, Aix-Marseille Université

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development