using-linear-agarose-channels-to-study-drosophila-larval-crawling

Using Linear Agarose Channels to Study <em>Drosophila</em> Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

University of Chicago

Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets.

An image-free opto-mechanical system for creating virtual environments and imaging neuronal activity in freely moving Caenorhabditis elegans.

Understanding rhythmic behavior at the developmental and genetic levels has important implications for neurobiology, medicine, evolution, and robotics. We studied rhythmic behavior--larval crawling--in the genetically and developmentally tractable organism, Drosophila melanogaster. We used narrow-diameter channels to constrain behavior to simple, rhythmic crawling. We quantified crawling at the organism, segment, and muscle levels. We showed that Drosophila larval crawling is made up of a series of periodic strides. Each stride consists of two phases. First, while most abdominal segments remain planted on the substrate, the head, tail, and gut translocate; this "visceral pistoning" moves the center of mass. The movement of the center of mass is likely powered by muscle contractions in the head and tail. Second, the head and tail anchor while a body wall wave moves each abdominal segment in the direction of the crawl. These two phases can be observed occurring independently in embryonic stages before becoming coordinated at hatching. During forward crawls, abdominal body wall movements are powered by simultaneous contraction of dorsal and ventral muscle groups, which occur concurrently with contraction of lateral muscles of the adjacent posterior segment. During reverse crawls, abdominal body wall movements are powered by phase-shifted contractions of dorsal and ventral muscles; and ventral muscle contractions occur concurrently with contraction of lateral muscles in the adjacent anterior segment. This work lays a foundation for use of Drosophila larva as a model system for studying the genetics and development of rhythmic behavior.

Characterization of Drosophila larval crawling at the level of organism, segment, and somatic body wall musculature.

&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x63CF;&#x8FF0; 5,000 GAL4 &#x884C;&#x4F7F;&#x7528;&#x5206;&#x5B50;&#x5B9A;&#x4E49;&#x7684;&#x72EC;&#x8054;&#x4F53;&#x76D1;&#x7BA1; DNA &#x63D2;&#x5165;&#x5230;&#x5355;&#x4E2A; attP &#x57FA;&#x56E0;&#x7EC4;&#x4F4D;&#x7F6E;&#x8FDB;&#x884C;&#x80DA;&#x80CE;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x6211;&#x4EEC;&#x8BB0;&#x5F55;&#x548C;&#x6CE8;&#x91CA;&#x7684;&#x6A21;&#x5F0F;&#x5728;&#x65E9;&#x671F;&#x80DA;&#x80CE;&#x795E;&#x7ECF;&#x53D1;&#x751F;&#x65F6;&#x8FBE;&#x5230;&#x9876;&#x5CF0;&#xFF0C;&#x548C;&#x65E7;&#x7684;&#x80DA;&#x80CE;&#x6709;&#x6781;&#x5927;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x591A;&#x6837;&#x6027;&#xFF0C;&#x5E76;&#x5EFA;&#x7ACB;&#x4E86;&#x7B2C;&#x4E00;&#x4E2A;&#x795E;&#x7ECF;&#x7535;&#x8DEF;&#x3002;&#x6211;&#x4EEC;&#x6CE8;&#x610F;&#x5230;&#xFF0C;&#x8EAB;&#x4F53;&#x4FA7;&#x58C1; (&#x808C;&#x8089;&#x3001; &#x611F;&#x89C9;&#x795E;&#x7ECF;&#x5143;&#x548C;&#x6C14;&#x7BA1;&#xFF09; &#x548C;&#x5185;&#x810F;&#x7B49;&#x5176;&#x4ED6;&#x7EC4;&#x7EC7;&#x4E2D;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x5173;&#x4E8E;&#x6210;&#x4EBA;&#x5927;&#x8111;&#x548C;&#x5E7C;&#x866B;&#x7684;&#x5F62;&#x8C61;&#x6027;&#x5149;&#x76D8;&#x548C;&#x7EFC;&#x5408;&#x7684;&#x6570;&#x636E;&#x96C6;&#x7684;&#x540C;&#x4F34;&#x8BBA;&#x6587;&#x62A5;&#x544A;&#x90FD;&#x53EF;&#x7528;&#x5728;&#x7EBF; (http://www.janelia.org/gal4-gen1)&#x3002;&#x6B64;&#x96C6;&#x5408;&#x7684; embryonically &#x8868;&#x793A; GAL4 &#x7EBF;&#x5C06;&#x5B9D;&#x8D35;&#x786E;&#x5B9A;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x5F62;&#x6001;&#x548C;&#x529F;&#x80FD;&#x3002;&#x5C0F;&#x7684;&#x5B50;&#x96C6;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x4E2D;&#x8868;&#x8FBE;&#x7684; 1,862 &#x7EBF; &#xFF08;&lt; 20/&#x6BB5;&#xFF09; &#x5C06;&#x8868;&#x5F81; interneuronal &#x591A;&#x6837;&#x6027;&#x4E0E;&#x529F;&#x80FD;&#xFF0C;&#x5C24;&#x5176;&#x662F;&#x5B9D;&#x8D35;&#xFF0C;&#x56E0;&#x4E3A;&#x867D;&#x7136; interneurons &#x5360;&#x6240;&#x6709;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x5927;&#x591A;&#x6570;&#xFF0C;&#x4F46;&#x5B83;&#x4EEC;&#x7684;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x8C31;&#x548C;&#x529F;&#x80FD;&#x4ECD;&#x51E0;&#x4E4E;&#x86EE;&#x8352;&#x4E4B;&#x5730;&#x3002;

&#x64CD;&#x7EB5;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x548C;&#x5206;&#x6790;&#x72EC;&#x8054;&#x4F53;&#x76D1;&#x7BA1;&#x6A21;&#x5757;&#x5728;&#x679C;&#x8747;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x4E2D;&#x7684;&#x8D44;&#x6E90;&#x3002;

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data setsÂ are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizingÂ interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.

A Resource for Manipulating Gene Expression and Analyzing cis-Regulatory Modules in the Drosophila CNS.

Nous d&eacute;crivons ici l&#39;expression embryonnaire du syst&egrave;me nerveux central de 5 000 lign&eacute;es GAL4 faite en utilisant l&#39;ADN de cis-r&eacute;gulation mol&eacute;culairement d&eacute;fini ins&eacute;r&eacute;e dans un emplacement de g&eacute;nomique attP unique. Nous documenter et annoter les sch&eacute;mas chez les embryons au d&eacute;but lorsque la neurogen&egrave;se est &agrave; son apog&eacute;e et chez les embryons &acirc;g&eacute;s o&ugrave; il y a une diversit&eacute; neuronale maximale et les premiers circuits neurones sont &eacute;tablis. Nous constatons d&#39;expression dans d&#39;autres tissus, comme la paroi lat&eacute;rale du corps (muscles, neurones sensoriels et trach&eacute;e) et les visc&egrave;res. Rapport de documents d&#39;accompagnement sur le cerveau adulte et larvaires disques imaginaux et les ensembles de donn&eacute;es int&eacute;gr&eacute;es sont disponibles en ligne (http://www.janelia.org/gal4-gen1). Cette collection de lignes de GAL4 embryonnaire exprim&eacute;s seront utile pour d&eacute;terminer la fonction et la morphologie neuronale. Les lignes 1 862 exprim&eacute;es en petits sous-ensembles de neurones (&lt; 20/segment) sera particuli&egrave;rement utile pour caract&eacute;riser la diversit&eacute; interneuronal et fonction, parce que m&ecirc;me si les interneurones constituent la majorit&eacute; de tous les neurones du syst&egrave;me nerveux central, leur profil d&#39;expression des g&egrave;nes et leurs fonctions demeurent pratiquement inexplor&eacute;es.

Une ressource pour manipuler l&#39;Expression des g&egrave;nes et analyse des Modules cis-r&eacute;glementaires dans le SNC de drosophile.

Hier beschreiben wir den embryonalen Zentralnervensystem Ausdruck von 5.000 GAL4-Linien unter Verwendung molekular definierter Cis-regulatorische DNA, die in einer einzigen AttP genomische Position eingef&uuml;gt. Wir dokumentieren und kommentieren die Muster in fr&uuml;hen Embryonen beim Neurogenese am H&ouml;hepunkt ist und in &auml;lteren Embryonen, wo maximale neuronale Vielfalt herrscht und die erste neuronale Schaltkreise sind etabliert. Wir beachten Sie Ausdruck in anderen Geweben, wie z. B. die seitliche Wand der K&ouml;rper (Muskel, sensorische Neuronen und Luftr&ouml;hre) und Eingeweide. Begleiter-Papiere-Bericht &uuml;ber die erwachsenen Gehirn und larval bildlichen Scheiben und die integrierte Daten-Sets sind online verf&uuml;gbar (http://www.janelia.org/gal4-gen1). Diese Sammlung von im Embryonialstadium exprimierten GAL4-Linien werden wertvolle zur Bestimmung neuronale Morphologie und Funktion. Die 1.862 Linien ausgedr&uuml;ckt in kleinen Teilmengen von Neuronen (&lt; 20/Segment) werden besonders wertvoll f&uuml;r die Charakterisierung von interneuronales Vielfalt und Funktion, denn obwohl Interneuronen die Mehrheit aller Neuronen des Zentralnervensystems umfassen, ihre Genexpressionsprofil und Funktion praktisch unerforscht bleiben.

Eine Ressource f&uuml;r die Bearbeitung von Genexpression und analysieren Cis-regulatorische Module im ZNS Drosophila.

Qui, descriviamo l&#39;espressione embrionale del sistema nervoso centrale di 5.000 righe di GAL4 realizzati utilizzando molecolarmente definito cis-regolatori DNA inserito in una posizione di genomic attP singolo. Abbiamo documento e annotare i modelli primi embrioni quando neurogenesi &egrave; al suo picco e in pi&ugrave; vecchi embrioni sono stabiliti i primi circuiti neurali e dove c&#39;&egrave; la massima diversit&agrave; neuronale. Notiamo l&#39;espressione in altri tessuti, come ad esempio la parete laterale del corpo (muscoli, neuroni sensoriali e trachea) e visceri. Relazione carte compagno sul cervello adulto e larvale dischi immaginale e gli insiemi di dati integrati sono disponibili online (http://www.janelia.org/gal4-gen1). Questa raccolta di embryonically espressi GAL4 linee sar&agrave; utile per determinare la funzione e morfologia neuronale. Le 1.862 linee espresse in piccoli sottoinsiemi di neuroni (&lt; 20/segmento) sar&agrave; particolarmente utile per caratterizzare la diversit&agrave; interneuronal e funzione, perch&eacute; anche se interneurons comprendono la maggior parte di tutti i neuroni del sistema nervoso centrale, il loro profilo di espressione genica e la funzione rimangono praticamente inesplorate.

Una risorsa per la manipolazione di espressione genica e analizzando i moduli cis-regolatori nel SNC della drosofila.

&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x5358;&#x4E00; attP &#x306E;&#x30B2;&#x30CE;&#x30E0;&#x5834;&#x6240;&#x306B;&#x633F;&#x5165;&#x5206;&#x5B50;&#x5B9A;&#x7FA9;&#x6E08;&#x307F; cis &#x898F;&#x5236; DNA &#x3092;&#x4F7F;&#x7528;&#x3057;&#x305F; 5,000 &#x306E; GAL4 &#x7DDA;&#x306E;&#x80DA;&#x306E;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x8868;&#x73FE;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x6587;&#x66F8;&#x5316;&#x3057;&#x3001;&#x795E;&#x7D4C;&#x65B0;&#x751F;&#x304C;&#x30D4;&#x30FC;&#x30AF;&#x6642;&#x306B;&#x3068;&#x304D;&#x306E;&#x521D;&#x671F;&#x80DA;&#x3068;&#x3088;&#x308A;&#x53E4;&#x3044;&#x80DA;&#x306E;&#x6975;&#x5927;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x591A;&#x69D8;&#x6027;&#x304C;&#x3042;&#x308A;&#x3001;&#x6700;&#x521D;&#x306E;&#x795E;&#x7D4C;&#x56DE;&#x8DEF;&#x304C;&#x78BA;&#x7ACB;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306B;&#x6CE8;&#x91C8;&#x3092;&#x4ED8;&#x3051;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x5F0F; &#xFF08;&#x7B4B;&#x8089;&#x3001;&#x611F;&#x899A;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3068;&#x6C17;&#x7BA1;&#xFF09; &#x306E;&#x5916;&#x5074;&#x306E;&#x4F53;&#x58C1;&#x3084;&#x5185;&#x81D3;&#x306A;&#x3069;&#x306E;&#x4ED6;&#x306E;&#x7D44;&#x7E54;&#x306B;&#x6CE8;&#x610F;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002;&#x30B3;&#x30F3;&#x30D1;&#x30CB;&#x30AA;&#x30F3; &#x30DA;&#x30FC;&#x30D1;&#x30FC; &#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x3001;&#x6210;&#x4EBA;&#x306E;&#x8133;&#x3068;&#x5E7C;&#x751F;&#x306E;&#x6210;&#x866B;&#x539F;&#x57FA;&#x3068;&#x7D71;&#x5408;&#x3055;&#x308C;&#x305F;&#x30C7;&#x30FC;&#x30BF; &#x30BB;&#x30C3;&#x30C8;&#x304C;&#x5229;&#x7528;&#x53EF;&#x80FD;&#x306A;&#x30AA;&#x30F3;&#x30E9;&#x30A4;&#x30F3; &#xFF08;http://www.janelia.org/gal4-gen1&#xFF09; &#x3067;&#x3059;&#x3002;&#x3053;&#x306E; embryonically &#x8868;&#x660E; GAL4 &#x884C;&#x306E;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x5F62;&#x614B;&#x3068;&#x6A5F;&#x80FD;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x8CB4;&#x91CD;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x3067;&#x8868;&#x660E;&#x3057;&#x305F; 1,862 &#x30E9;&#x30A4;&#x30F3; (&lt; 20/&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;) &#x4ECB;&#x5728;&#x795E;&#x7D4C;&#x3059;&#x3079;&#x3066;&#x306E;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x5927;&#x591A;&#x6570;&#x3092;&#x69CB;&#x6210;&#x3059;&#x308B;&#x304C;&#x3001;&#x5F7C;&#x3089;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3068;&#x95A2;&#x6570;&#x4E8B;&#x5B9F;&#x4E0A;&#x672A;&#x8E0F;&#x306E;&#x307E;&#x307E;&#x306E;&#x3067;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x591A;&#x69D8;&#x6027;&#x3068;&#x6A5F;&#x80FD;&#x3001;&#x7279;&#x6027;&#x8A55;&#x4FA1;&#x306B;&#x7279;&#x306B;&#x4FA1;&#x5024;&#x304C;&#x3042;&#x308B;&#x3067;&#x3057;&#x3087;&#x3046;&#x3002;

&#x64CD;&#x4F5C;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x304A;&#x3088;&#x3073;&#x5206;&#x6790;&#x30B7;&#x30B9;&#x8ABF;&#x7BC0;&#x30E2;&#x30B8;&#x30E5;&#x30FC;&#x30EB; &#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x306E;&#x305F;&#x3081;&#x306E;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3067;&#x3059;&#x3002;

&#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xB294; &#xB2E8;&#xC77C; attP &#xAC8C;&#xB188; &#xC704;&#xCE58;&#xC5D0; &#xC0BD;&#xC785; &#xD558;&#xB294; &#xBD84;&#xC790;&#xB85C; &#xC815;&#xC758; cis &#xADDC;&#xC815; DNA&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB9CC;&#xB4E0; 5000 GAL4 &#xB77C;&#xC778;&#xC758; &#xBC30;&#xC544; &#xC911;&#xCD94; &#xC2DD; &#xC124;&#xBA85;. &#xC6B0;&#xB9AC;&#xB294; &#xBB38;&#xC11C;&#xD654; &#xD558; &#xACE0; &#xC8FC;&#xC11D;&#xC744; &#xB2EC; &#xB54C; &#xC131;&#xCCB4;&#xB294; &#xADF8; &#xC808;&#xC815;&#xC5D0; &#xC774;&#xB978; &#xBC30;&#xC544; &#xBC0F; &#xC774;&#xC804; &#xBC30;&#xC544;&#xB294; &#xCD5C;&#xB300;&#xD55C; &#xC2E0;&#xACBD; &#xB2E4;&#xC591;&#xC131; &#xBC0F; &#xCCAB; &#xBC88;&#xC9F8; &#xC2E0;&#xACBD; &#xD68C;&#xB85C; &#xC124;&#xB9BD; &#xD558;&#xB294; &#xD328;&#xD134;. &#xC6B0;&#xB9AC;&#xB294; &#xC2DD; &#xCE21;&#xBA74; &#xBC14;&#xB514; &#xBCBD; (&#xADFC;&#xC721;, &#xAC10;&#xAC01; &#xC2E0;&#xACBD; &#xBC0F; &#xAE30;&#xAD00;&#xC9C0;) &#xBC0F; &#xB0B4;&#xC7A5; &#xB4F1; &#xB2E4;&#xB978; &#xC870;&#xC9C1;&#xC5D0; &#xC8FC;&#xC758;. &#xC131;&#xC778; &#xB450;&#xB1CC;&#xC640; &#xC560;&#xBC8C;&#xB808; imaginal &#xB514;&#xC2A4;&#xD06C;&#xC640; &#xD1B5;&#xD569;&#xB41C; &#xB370;&#xC774;&#xD130; &#xC9D1;&#xD569; &#xB3D9;&#xBC18;&#xC790; &#xB17C;&#xBB38; &#xBCF4;&#xACE0;&#xC11C;&#xB294; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xC628;&#xB77C;&#xC778; (http://www.janelia.org/gal4-gen1). Embryonically &#xD45C;&#xD604;&#xB41C; GAL4 &#xB77C;&#xC778;&#xC758;&#xC774; &#xCEEC;&#xB809;&#xC158; &#xC2E0;&#xACBD; &#xD615;&#xD0DC;&#xC640; &#xAE30;&#xB2A5;&#xC744; &#xACB0;&#xC815; &#xD558;&#xB294; &#xB370; &#xB3C4;&#xC6C0;&#xC774; &#xB420; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;. &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC791;&#xC740; &#xD558;&#xC704; &#xC9D1;&#xD569; &#xB2E8;&#xC704;&#xB85C; 1862 &#xB77C;&#xC778; (&lt; 20/&#xC138;&#xADF8;&#xBA3C;&#xD2B8;) &#xC218; &#xBAA8;&#xB4E0; &#xC911;&#xCD94; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xB300;&#xB2E4;&#xC218;&#xB97C; &#xAD6C;&#xC131;, &#xD558;&#xC9C0;&#xB9CC; &#xADF8;&#xB4E4;&#xC758; &#xC9C4; &#xC2DD; &#xD504;&#xB85C;&#xD544;&#xACFC; &#xAE30;&#xB2A5; &#xAC70;&#xC758; &#xBBF8;&#xAC1C;&#xCC99; &#xB0A8;&#xC544; &#xC788;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xD2B9;&#xD788; interneuronal &#xB2E4;&#xC591; &#xC131;&#xACFC; &#xAE30;&#xB2A5;, &#xD2B9;&#xC131;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xAC00;&#xCE58;&#xAC00; &#xC788;&#xC744; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;.

&#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604; &#xC870;&#xC791; &#xBC0F; &#xBD84;&#xC11D; Drosophila CNS&#xC5D0; cis &#xADDC;&#xC815; &#xBAA8;&#xB4C8;&#xC744; &#xC704;&#xD55C; &#xC790;&#xC6D0;.

Aqu&iacute;, describimos la expresi&oacute;n embrionaria del sistema nervioso central de 5.000 l&iacute;neas de GAL4 constituidas ADN cis-regulaci&oacute;n molecular definida insertado en una localizaci&oacute;n gen&oacute;mica attP solo. Documentamos y anotar los patrones en embriones tempranos cuando neurog&eacute;nesis est&aacute; en su apogeo y en mayores embriones donde hay m&aacute;xima diversidad neuronal y se establecen los primeros circuitos neuronales. Tomamos nota de expresi&oacute;n en otros tejidos, como la pared lateral del cuerpo (m&uacute;sculo, neuronas sensoriales y tr&aacute;quea) y v&iacute;sceras. Informe de papeles de compa&ntilde;ero en el cerebro adulto y larvarios discos imaginal y los conjuntos de datos integrados est&aacute;n disponibles en l&iacute;nea (http://www.janelia.org/gal4-gen1). Esta colecci&oacute;n de manera embrional expresas GAL4 l&iacute;neas ser&aacute;n valiosa para determinar la funci&oacute;n y morfolog&iacute;a neuronal. Las l&iacute;neas de 1.862 expresadas en peque&ntilde;os subconjuntos de neuronas (&lt; 20/segmento) ser&aacute; especialmente valioso para la caracterizaci&oacute;n de la diversidad interneuronal y funci&oacute;n, porque aunque interneuronas constituyen la mayor&iacute;a de todas las neuronas del sistema nervioso central, su perfil de expresi&oacute;n g&eacute;nica y la funci&oacute;n permanecen pr&aacute;cticamente inexplorados.

Un recurso para manipulaci&oacute;n de expresi&oacute;n g&eacute;nica y m&oacute;dulos de an&aacute;lisis cis-reguladoras del sistema nervioso de Drosophila.

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.

Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.

Bilaterally symmetric motor patterns--those in which left-right pairs of muscles contract synchronously and with equal amplitude (such as breathing, smiling, whisking, and locomotion)--are widespread throughout the animal kingdom. Yet, surprisingly little is known about the underlying neural circuits. We performed a thermogenetic screen to identify neurons required for bilaterally symmetric locomotion in Drosophila larvae and identified the evolutionarily conserved Even-skipped(+) interneurons (Eve/Evx). Activation or ablation of Eve(+) interneurons disrupted bilaterally symmetric muscle contraction amplitude, without affecting the timing of motor output. Eve(+) interneurons are not rhythmically active and thus function independently of the locomotor CPG. GCaMP6 calcium imaging of Eve(+) interneurons in freely moving larvae showed left-right asymmetric activation that correlated with larval behavior. TEM reconstruction of Eve(+) interneuron inputs and outputs showed that the Eve(+) interneurons are at the core of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction.

Even-Skipped(+) Interneurons Are Core Components of a Sensorimotor Circuit that Maintains Left-Right Symmetric Muscle Contraction Amplitude.

Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines-chosen for sparse neuronal expression-to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program.

Ellie S. Heckscher

Committee on Development,
Regeneration,
and Stem Cell Biology,
Department of Molecular Genetics and Cell Biology,
Committee on Development, Regeneration, and Stem Cell Biology

University of Chicago

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Ellie S. Heckscher

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Committee on Development,Regeneration,and Stem Cell Biology,Department of Molecular Genetics and Cell Biology,Committee on Development, Regeneration, and Stem Cell Biology

University of Chicago

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Committee on Development,
Regeneration,
and Stem Cell Biology,
Department of Molecular Genetics and Cell Biology,
Committee on Development, Regeneration, and Stem Cell Biology