drosophila-courtship-conditioning-as-a-measure-of-learning-and-memory

<em>Drosophila</em> Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Howard Hughes Medical Institute

Netrins.

Crecimiento del ax&oacute;n a trav&eacute;s de la l&iacute;nea media de Drosophila requiere Comm para regular a la baja Robo, el receptor para la ranura del midline repelente. Aqu&iacute; mostramos que comm es necesaria en las neuronas, no en las c&eacute;lulas de la l&iacute;nea media como se pensaba y se expresa espec&iacute;ficamente y transitoriamente en las neuronas commissural. COMM act&uacute;a como un receptor de clasificaci&oacute;n para Robo, desvi&aacute;ndola de lo sint&eacute;tico a la v&iacute;a endoc&iacute;ticas final. Un motivo LPSY citopl&aacute;smico conservado se requiere endosomal clasificaci&oacute;n de Comm in vitro y Comm para regular a la baja Robo y promover el cruce de la l&iacute;nea media en vivo. Tr&aacute;nsito de ax&oacute;n en la l&iacute;nea media del CNS as&iacute; es controlado por el tr&aacute;fico intracelular de los receptores de la direcci&oacute;n de Robo, que a su vez depende de la expresi&oacute;n precisamente regulada de la Comm clasificaci&oacute;n del receptor.

COMM incluye robo la orientaci&oacute;n del ax&oacute;n de control en la l&iacute;nea media de Drosophila.

&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x6B63;&#x4E2D;&#x7DDA;&#x3067;&#x8EF8;&#x7D22;&#x6210;&#x9577;&#x30EC;&#x30BB;&#x30D7;&#x30BF;&#x30FC; &#x30ED;&#x30DC;&#x3001;&#x6B63;&#x4E2D;&#x7DDA;&#x64A5;&#x306E;&#x30B9;&#x30EA;&#x30C3;&#x30C8;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x306B;&#x901A;&#x4FE1;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x901A;&#x4FE1;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3001;&#x4EE5;&#x524D;&#x306F;&#x601D;&#x3063;&#x3066;&#x3001;&#x6B63;&#x4E2D;&#x7DDA;&#x7D30;&#x80DE;&#x3067;&#x5177;&#x4F53;&#x7684;&#x306B;&#x306F;&#x3001;&#x4E00;&#x6642;&#x7684;&#x4EA4;&#x9023;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x73FE;&#x306F;&#x306A;&#x3044;&#x3092;&#x793A;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002;&#x901A;&#x4FE1;&#x3068;&#x3057;&#x3066;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x53D7;&#x5BB9;&#x4F53;&#x5408;&#x6210;&#x304B;&#x3089;&#x5F8C;&#x534A;&#x306E;&#x30A8;&#x30F3;&#x30C9;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x7D4C;&#x8DEF;&#x306B;&#x307E;&#x304E;&#x308C;&#x3082;&#x306A;&#x304F;&#x30ED;&#x30DC;&#x306E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x307E;&#x3059;&#x3002;&#x4FDD;&#x5B58;&#x306E;&#x7D30;&#x80DE;&#x8CEA; LPSY &#x30E2;&#x30C1;&#x30FC;&#x30D5; &#x30A8;&#x30F3;&#x30C9;&#x30BD;&#x30FC;&#x30E0;&#x901A;&#x4FE1; in vitro &#x3067;&#x306E;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x3068;&#x901A;&#x4FE1;&#x30EC;&#x30BB;&#x30D7;&#x30BF;&#x30FC; &#x30ED;&#x30DC;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3001;in vivo &#x3067;&#x6B63;&#x4E2D;&#x7DDA;&#x306E;&#x4EA4;&#x5DEE;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x306E;&#x6B63;&#x4E2D;&#x7DDA;&#x306E;&#x8EF8;&#x7D22;&#x30C8;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30AF;&#x306F;&#x3001;&#x3053;&#x306E;&#x3088;&#x3046;&#x306B;&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x901A;&#x4FE1;&#x306E;&#x6B63;&#x78BA;&#x306B;&#x898F;&#x5236;&#x306E;&#x5F0F;&#x306F;&#x9806;&#x756A;&#x306B;&#x30ED;&#x30DC; &#x30AC;&#x30A4;&#x30C0;&#x30F3;&#x30B9;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x8F38;&#x9001;&#x306B;&#x3088;&#x3063;&#x3066;&#x5236;&#x5FA1;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;

&#x901A;&#x4FE1;&#x5236;&#x5FA1;&#x8EF8;&#x7D22;&#x30AC;&#x30A4;&#x30C0;&#x30F3;&#x30B9; &#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x6B63;&#x4E2D;&#x7DDA;&#x3067;&#x30ED;&#x30DC;&#x3092;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x307E;&#x3059;&#x3002;

Drosophila &#xC911;&#xAC04;&#xC5D0; &#xAC78;&#xCCD0; &#xCD95; &#xC0AD; &#xC131;&#xC7A5; downregulate&#xB85C; &#xBCF4;, &#xC911;&#xAC04; &#xAD6C; &#xCDA9; &#xC81C; &#xC2AC;&#xB9BF;&#xC5D0; &#xB300; &#xD55C; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xD1B5;&#xC2E0; &#xD574;&#xC57C;&#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC5EC;&#xAE30; &#xD1B5;&#xC2E0; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0; &#xD544;&#xC694;&#xD55C;, &#xD45C;&#xD604; &#xD558;&#xC9C0; &#xC774;&#xC804;&#xC5D0; &#xC0DD;&#xAC01; &#xD558;&#xB294; &#xC911;&#xAC04; &#xC120; &#xC138;&#xD3EC;&#xC5D0; &#xB41C; &#xAD6C;&#xCCB4;&#xC801;&#xC73C;&#xB85C; &#xADF8;&#xB9AC;&#xACE0; transiently commissural &#xC2E0;&#xACBD; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xD1B5;&#xC2E0;&#xC73C;&#xB85C; &#xBCF4;, &#xD6C4;&#xBC18; endocytic &#xD1B5;&#xB85C;&#xC5D0; &#xD569;&#xC131;&#xC5D0;&#xC11C; &#xC6B0;&#xD68C;&#xC5D0; &#xB300; &#xD55C; &#xC815;&#xB82C; &#xC218;&#xC6A9; &#xCCB4;&#xB85C; &#xC11C; &#xC5ED;&#xD560;&#xC744; &#xD569;&#xB2C8;&#xB2E4;. &#xBCF4;&#xC874;&#xB41C; &#xC138;&#xD3EC;&#xC9C8; LPSY &#xBAA8;&#xD2F0;&#xBE0C; endosomal &#xD1B5;&#xC2E0; &#xCCB4; &#xC678;&#xC758; &#xC815;&#xB82C; &#xD558; &#xACE0; &#xD1B5;&#xC2E0; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694;&#xD55C; downregulate&#xB85C; &#xBCF4;&#xC640; &#xC911;&#xAC04; &#xC120; &#xD6A1;&#xB2E8; vivo&#xC5D0;&#xC11C; &#xD64D;&#xBCF4;. CNS &#xC911;&#xAC04;&#xC5D0;&#xC11C; &#xCD95; &#xC0AD; &#xD2B8;&#xB798;&#xD53D;&#xC740; &#xB530;&#xB77C;&#xC11C; &#xC138;&#xD3EC;&#xB0B4; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xBD84;&#xB958; &#xD558;&#xB294; &#xD1B5;&#xC2E0;&#xC758; &#xC815;&#xD655; &#xD558; &#xAC8C; &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; &#xC2DD;&#xC5D0; &#xB530;&#xB77C; &#xCC28;&#xB840;&#xB85C; &#xBCF4; &#xC9C0;&#xCE68; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xC758;&#xD574; &#xC81C;&#xC5B4; &#xB429;&#xB2C8;&#xB2E4;.

&#xD1B5;&#xC2E0; Drosophila &#xC911;&#xAC04;&#xC5D0;&#xC11C; &#xC81C;&#xC5B4; &#xCD95; &#xC0AD; &#xC9C0;&#xCE68;&#xC744;&#xB85C; &#xBCF4;&#xB97C; &#xC815;&#xB82C;&#xD569;&#xB2C8;&#xB2E4;.

&#x8F74;&#x7A81;&#x751F;&#x957F;&#x5728;&#x679C;&#x8747;&#x4E2D;&#x7EBF;&#x4E4B;&#x95F4;&#x9700;&#x8981;&#x56FD;&#x9645;&#x4FE1;&#x606F;&#x901A;&#x4FE1;&#x5C55;&#x89C8;&#x4F1A;&#x5411;&#x4E0B;&#x8C03; Robo&#xFF0C;&#x4E2D;&#x7EBF;&#x9A71;&#x868A;&#x5242;&#x7F1D;&#x9699;&#x7684;&#x53D7;&#x4F53;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x8FD9;&#x91CC;&#x5C55;&#x793A;&#x56FD;&#x9645;&#x4FE1;&#x606F;&#x901A;&#x4FE1;&#x5C55;&#x89C8;&#x4F1A;&#x795E;&#x7ECF;&#x5143;&#x6240;&#x9700;&#xFF0C;&#x4E0D;&#x5728;&#x4E2D;&#x7EBF;&#x7EC6;&#x80DE;&#x50CF;&#x4EE5;&#x524D;&#x8BA4;&#x4E3A;&#xFF0C;&#x548C;&#x8FD9;&#x5B83;&#x8868;&#x793A;&#x5177;&#x4F53;&#x548C;&#x77AC;&#x6001;&#x9AD3;&#x540E;&#x8FDE;&#x5408;&#x795E;&#x7ECF;&#x5143;&#x3002;&#x56FD;&#x9645;&#x4FE1;&#x606F;&#x901A;&#x4FE1;&#x5C55;&#x89C8;&#x4F1A;&#x4F5C;&#x4E3A; Robo&#xFF0C;&#x5B83;&#x5F15;&#x5411;&#x665A;&#x541E;&#x9014;&#x5F84;&#x7684;&#x7EFC;&#x5408;&#x6392;&#x5E8F;&#x7684;&#x53D7;&#x4F53;&#x3002;&#x9700;&#x8981; endosomal &#x6392;&#x5E8F;&#x7684; Comm &#x4F53;&#x5916;&#x548C;&#x56FD;&#x9645;&#x4FE1;&#x606F;&#x901A;&#x4FE1;&#x5C55;&#x89C8;&#x4F1A;&#x5411;&#x4E0B;&#x8C03; Robo &#x5B88;&#x6052;&#x7EC6;&#x80DE;&#x8D28; LPSY &#x6BCD;&#x9898;&#xFF0C;&#x4FC3;&#x8FDB;&#x4F53;&#x5185;&#x7684;&#x4E2D;&#x7EBF;&#x8FC7;&#x5883;&#x3002;CNS &#x4E2D;&#x7EBF;&#x7684;&#x8F74;&#x7A81;&#x4EA4;&#x901A;&#x56E0;&#x6B64;&#x53D7;&#x63A7;&#x5236; Robo &#x6307;&#x5BFC;&#x53D7;&#x4F53;&#xFF0C;&#x53C8;&#x53D6;&#x51B3;&#x4E8E;&#x6392;&#x5E8F;&#x53D7;&#x4F53;&#x7684; Comm &#x7684;&#x7CBE;&#x786E;&#x8C03;&#x8282;&#x8868;&#x8FBE;&#x7684;&#x7EC6;&#x80DE;&#x5185;&#x8D29;&#x5356;&#x3002;

&#x56FD;&#x9645;&#x4FE1;&#x606F;&#x901A;&#x4FE1;&#x5C55;&#x89C8;&#x4F1A;&#x5728;&#x679C;&#x8747;&#x4E2D;&#x7EBF;&#x63A7;&#x5236;&#x8F74;&#x7A81;&#x5BFC;&#x5411;&#x5230;&#x6392;&#x5E8F; robo&#x3002;

Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. We show here that comm is required in neurons, not in midline cells as previously thought, and that it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor.

Comm sorts robo to control axon guidance at the Drosophila midline.

Croissance axonale dans l&#39;ensemble de la ligne m&eacute;diane de drosophile exige Comm downregulate Robo, le r&eacute;cepteur pour la fente m&eacute;diane r&eacute;pulsif. Nous montrons ici que la comm est requis dans les neurones, pas dans les cellules de la ligne m&eacute;diane que l&#39;on croyait et qu&#39;il est exprim&eacute; sp&eacute;cifiquement et de fa&ccedil;on transitoire dans les neurones commissurale. Comm agit comme un r&eacute;cepteur tri pour Robo, d&eacute;tourner de la synth&egrave;se de la voie de l&#39;endocytose tardive. Un motif conserv&eacute; de LPSY cytoplasmique doit pour endosomale tri des Comm in vitro et Comm downregulate Robo et promouvoir le franchissement de la ligne m&eacute;diane in vivo. Le trafic sur la ligne m&eacute;diane du CNS AXON est ainsi contr&ocirc;l&eacute; par le trafic intracellulaire du r&eacute;cepteur Robo orientation, qui d&eacute;pend &agrave; son tour l&#39;expression pr&eacute;cis&eacute;ment r&eacute;gul&eacute;e de la Comm tri du r&eacute;cepteur.

Comm sortes robo &agrave; guidage axonal contr&ocirc;le &agrave; la ligne m&eacute;diane de drosophile.

Axon Zuw&auml;chse in der Taufliege-Mittellinie erfordert Comm gulieren Robo, den Rezeptor f&uuml;r die Mittellinie abweisend Slit. Hier zeigen wir, dass Comm in Neuronen erforderlich ist, nicht in Mittellinie Zellen als bisher angenommen, und dass es ist spezifisch und vor&uuml;bergehend in kommissurale Neuronen exprimiert. Comm fungiert als ein Sortier-Rezeptor f&uuml;r Robo, es aus dem synthetischen umgeleitet, um der sp&auml;ten endocytic Weg. Konservierte zellplasmatisches LPSY Motiv ist verpflichtet f&uuml;r Endosomen Sortierung von in-vitro-Comm und Comm gulieren Robo und Mittellinie Kreuzung in-vivo f&ouml;rdern. Axon-Verkehr bei der CNS-Mittellinie wird somit durch intrazellul&auml;re Menschenhandel des Robo-Anleitung-Rezeptors, die wiederum abh&auml;ngig von der Comm sortieren Rezeptor genau geregelten Ausdruck gesteuert.

Comm sortiert Robo Steuerelement Axon Guidance der Drosophila-Mittellinie.

Assone crescita attraverso la linea mediana della drosofila richiede Comm a sottoregola Robo, il recettore per la fessura mediana repellente. Mostriamo qui che comm &egrave; necessaria nei neuroni, non nelle cellule della linea mediana come precedentemente pensate e che &egrave; espressa in modo specifico e transitoriamente in neuroni commissural. Comm agisce come un recettore ordinamento per Robo, deviando da sintetica del percorso endocitico tardo. Un motivo LPSY citoplasmatico conservato &egrave; richiesto per endosomal ordinamento di Comm in vitro e Comm sottoregola Robo e promuovere l&#39;attraversamento della linea mediana in vivo. Traffico assone presso la linea mediana CNS &egrave; cos&igrave; controllato da traffico intracellulare del recettore guida Robo, che dipende a sua volta l&#39;espressione precisamente regolamentato di Comm ordinamento del recettore.

Comm ordina robo per controllo assone guida presso la linea mediana della drosofila.

Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.

Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons.

Commissureless (Comm) controls axon guidance across the Drosophila melanogaster midline by regulating surface levels of Robo, the receptor for the midline repellent Slit. Two different models have been proposed for how Comm regulates Robo: a 'sorting' model and a 'clearance' model, both based on studies using heterologous cells in vitro. Here, we test these two models in vivo. We establish a genetic rescue assay for Comm, and use this assay to show that midline crossing does not require the presence of Comm in midline cells, as proposed by the clearance model. Moreover, by monitoring the trafficking of a Robo-green fluorescent protein (GFP) fusion in living embryos, we demonstrate that Comm prevents the delivery of Robo-GFP to the growth cone, as predicted by the sorting model. It has also been suggested that Comm must be ubiquitinated by the Nedd4 ubiquitin ligase. We show here, however, that ubiquitination of Comm is not required for its function in vitro or in vivo, and that Nedd4 is unlikely to function in axon guidance at the midline.

Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo.

Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.

A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.

Both long-term behavioral memory and synaptic plasticity require protein synthesis, some of which may occur locally at specific synapses. Cytoplasmic polyadenylation element-binding (CPEB) proteins are thought to contribute to the local protein synthesis that underlies long-term changes in synaptic efficacy, but a role has not been established for them in the formation of long-term behavioral memory. We found that the Drosophila melanogaster CPEB protein Orb2 is acutely required for long-term conditioning of male courtship behavior. Deletion of the N-terminal glutamine-rich region of Orb2 resulted in flies that were impaired in their ability to form long-term, but not short-term, memory. Memory was restored by expressing Orb2 selectively in fruitless (fru)-positive gamma neurons of the mushroom bodies and by providing Orb2 function in mushroom bodies only during and shortly after training. Our data thus demonstrate that a CPEB protein is important in long-term memory and map the molecular, spatial and temporal requirements for its function in memory formation.

Function of the Drosophila CPEB protein Orb2 in long-term courtship memory.

Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.

Systematic genetic analysis of muscle morphogenesis and function in Drosophila.

The epigenetic modification of chromatin structure and its effect on complex neuronal processes like learning and memory is an emerging field in neuroscience. However, little is known about the "writers" of the neuronal epigenome and how they lay down the basis for proper cognition. Here, we have dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT), a member of a conserved protein family that methylates histone 3 at lysine 9 (H3K9). EHMT is widely expressed in the nervous system and other tissues, yet EHMT mutant flies are viable. Neurodevelopmental and behavioral analyses identified EHMT as a regulator of peripheral dendrite development, larval locomotor behavior, non-associative learning, and courtship memory. The requirement for EHMT in memory was mapped to 7B-Gal4 positive cells, which are, in adult brains, predominantly mushroom body neurons. Moreover, memory was restored by EHMT re-expression during adulthood, indicating that cognitive defects are reversible in EHMT mutants. To uncover the underlying molecular mechanisms, we generated genome-wide H3K9 dimethylation profiles by ChIP-seq. Loss of H3K9 dimethylation in EHMT mutants occurs at 5% of the euchromatic genome and is enriched at the 5' and 3' ends of distinct classes of genes that control neuronal and behavioral processes that are corrupted in EHMT mutants. Our study identifies Drosophila EHMT as a key regulator of cognition that orchestrates an epigenetic program featuring classic learning and memory genes. Our findings are relevant to the pathophysiological mechanisms underlying Kleefstra Syndrome, a severe form of intellectual disability caused by mutations in human EHMT1, and have potential therapeutic implications. Our work thus provides novel insights into the epigenetic control of cognition in health and disease.

Epigenetic regulation of learning and memory by Drosophila EHMT/G9a.

Learning through trial-and-error interactions allows animals to adapt innate behavioural ‘rules of thumb’ to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.

Dopamine neurons modulate pheromone responses in Drosophila courtship learning.

Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist.

Drosophila CPEB Orb2A mediates memory independent of Its RNA-binding domain.

NDST1 was recently proposed as a candidate gene for autosomal recessive intellectual disability in two families. It encodes a bifunctional GlcNAc N-deacetylase/N-sulfotransferase with important functions in heparan sulfate biosynthesis. In mice, Ndst1 is crucial for embryonic development and homozygous null mutations are perinatally lethal. We now report on two additional unrelated families with homozygous missense NDST1 mutations. All mutations described to date predict the substitution of conserved amino acids in the sulfotransferase domain, and mutation modeling predicts drastic alterations in the local protein conformation. Comparing the four families, we noticed significant overlap in the clinical features, including both demonstrated and apparent intellectual disability, muscular hypotonia, epilepsy, and postnatal growth deficiency. Furthermore, in Drosophila, knockdown of sulfateless, the NDST ortholog, impairs long-term memory, highlighting its function in cognition. Our data confirm NDST1 mutations as a cause of autosomal recessive intellectual disability with a distinctive phenotype, and support an important function of NDST1 in human development.

NDST1 missense mutations in autosomal recessive intellectual disability.

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.

Identification of genes that promote or inhibit olfactory memory formation in Drosophila.

To adapt to an ever-changing environment, animals consolidate some, but not all, learning experiences to long-term memory. In mammals, long-term memory consolidation often involves neural pathway reactivation hours after memory acquisition. It is not known whether this delayed-reactivation schema is common across the animal kingdom or how information is stored during the delay period. Here, we show that, during courtship suppression learning, Drosophila exhibits delayed long-term memory consolidation. We also show that the same class of dopaminergic neurons engaged earlier in memory acquisition is also both necessary and sufficient for delayed long-term memory consolidation. Furthermore, we present evidence that, during learning, the translational regulator Orb2A tags specific synapses of mushroom body neurons for later consolidation. Consolidation involves the subsequent recruitment of Orb2B and the activity-dependent synthesis of CaMKII. Thus, our results provide evidence for the role of a neuromodulated, synapse-restricted molecule bridging memory acquisition and long-term memory consolidation in a learning animal.

Synaptic Orb2A Bridges Memory Acquisition and Late Memory Consolidation in Drosophila.

Localized protein translation is critical in many biological contexts, particularly in highly polarized cells, such as neurons, to regulate gene expression in a spatiotemporal manner. The cytoplasmic polyadenylation element-binding (CPEB) family of RNA-binding proteins has emerged as a key regulator of mRNA transport and local translation required for early embryonic development, synaptic plasticity, and long-term memory (LTM). Drosophila Orb and Orb2 are single members of the CPEB1 and CPEB2 subfamilies of the CPEB proteins, respectively. At present, the identity of the mRNA targets they regulate is not fully known, and the binding specificity of the CPEB2 subfamily is a matter of debate. Using transcriptome-wide UV cross-linking and immunoprecipitation, we define the mRNA-binding sites and targets of Drosophila CPEBs. Both Orb and Orb2 bind linear cytoplasmic polyadenylation element-like sequences in the 3' UTRs of largely overlapping target mRNAs, with Orb2 potentially having a broader specificity. Both proteins use their RNA-recognition motifs but not the Zinc-finger region for RNA binding. A subset of Orb2 targets is translationally regulated in cultured S2 cells and fly head extracts. Moreover, pan-neuronal RNAi knockdown of these targets suggests that a number of these targets are involved in LTM. Our results provide a comprehensive list of mRNA targets of the two CPEB proteins in Drosophila, thus providing insights into local protein synthesis involved in various biological processes, including LTM.

Article	Year
Netrins. Current biology : CB\| PubMed ID: 11882300	2002
Comm sorts robo to control axon guidance at the Drosophila midline. Cell\| PubMed ID: 12202032	2002
Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons. Genes & development\| PubMed ID: 15342493	2004
Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo. Nature neuroscience\| PubMed ID: 15657595	2005
A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature\| PubMed ID: 17625558	2007
Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nature neuroscience\| PubMed ID: 17965711	2007
Systematic genetic analysis of muscle morphogenesis and function in Drosophila. Nature\| PubMed ID: 20220848	2010
Epigenetic regulation of learning and memory by Drosophila EHMT/G9a. PLoS biology\| PubMed ID: 21245904	2011
Dopamine neurons modulate pheromone responses in Drosophila courtship learning. Nature\| PubMed ID: 22902500	2012
Drosophila CPEB Orb2A mediates memory independent of Its RNA-binding domain. Neuron\| PubMed ID: 23083740	2012
NDST1 missense mutations in autosomal recessive intellectual disability. American journal of medical genetics. Part A\| PubMed ID: 25125150	2014
Identification of genes that promote or inhibit olfactory memory formation in Drosophila. Genetics\| PubMed ID: 25644700	2015
Synaptic Orb2A Bridges Memory Acquisition and Late Memory Consolidation in Drosophila. Cell reports\| PubMed ID: 26095367	2015
RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27791065	2016

Krystyna Keleman

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