A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Washington University School of Medicine

&#x6211;&#x4EEC;&#x8BC1;&#x660E;&#x8BE5;&#x679C;&#x8747;&#x51EF;&#x666E;&#x83B1;&#x7279; (capt) &#x540C;&#x7CFB;&#x7269;&#x7684;&#x817A;&#x82F7;&#x9178;&#x73AF;&#x5316;&#x9176;&#x76F8;&#x5173;&#x86CB;&#x767D;&#x7684;&#x7ED1;&#x5B9A;&#x548C;&#x89C4;&#x7BA1;&#x5728;&#x9175;&#x6BCD;&#x4E2D;&#x7684;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x3001; Abl &#x679C;&#x8747;&#x7EC6;&#x80DE;&#xFF0C;&#x8FD9;&#x8868;&#x660E;&#x4F53;&#x5185;&#x7684;&#x529F;&#x80FD;&#x5173;&#x7CFB;&#x76F8;&#x5173;&#x8054;&#x3002;&#x6211;&#x4EEC;&#x627E;&#x5230;&#x9C81;&#x68D2;&#x548C;&#x7279;&#x5B9A;&#x9057;&#x4F20;&#x76F8;&#x4E92;&#x4F5C;&#x7528; capt &#x548C; Abl &#x5728;&#x90A3;&#x91CC;&#x751F;&#x957F;&#x9525;&#x9A71;&#x868A;&#x4E2D;&#x7EBF;&#x9009;&#x62E9;&#x70B9;&#x7F1D;&#x529F;&#x80FD;&#x6765;&#x9650;&#x5236;&#x8F74;&#x7A81;&#x8FC7;&#x5883;&#x3002;Capt &#x548C;&#x7F1D;&#x4E4B;&#x95F4;&#x7684;&#x9057;&#x4F20;&#x4EA4;&#x4E92;&#x652F;&#x6301; Capt &#x548C; Abl &#x5176;&#x4E2D;&#x62D2;&#x54CD;&#x5E94;&#x7684;&#x4E00;&#x90E8;&#x5206;&#x4F5C;&#x4E3A;&#x534F;&#x4F5C;&#x5904;&#x7406;&#x6A21;&#x578B;&#x3002;&#x7531; capt &#x548C; Abl &#x663E;&#x793A;&#x591A;&#x4E2A;&#x5BB6;&#x5EAD;&#x6210;&#x5458;&#x7684;&#x56DE;&#x65CB;&#x5904;&#x7684;&#x53D7;&#x4F53;&#x4E0E;&#x9057;&#x4F20;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x63D0;&#x4F9B;&#x8FDB;&#x4E00;&#x6B65;&#x7684;&#x652F;&#x6301;&#xFF0C;&#x8FD9;&#x79CD;&#x6A21;&#x5F0F;&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x786E;&#x5B9A;&#x51EF;&#x666E;&#x83B1;&#x7279;&#x4F5C;&#x4E3A;&#x6307;&#x5BFC;&#x4FE1;&#x53F7;&#x6302;&#x94A9;&#x8C03;&#x8282;&#x7EC6;&#x80DE;&#x9AA8;&#x67B6;&#x52A8;&#x529B;&#x5B66;&#x7684;&#x65B0;&#x5174;&#x901A;&#x8DEF;&#x7684;&#x4E00;&#x90E8;&#x5206;&#xFF0C;&#x5E76;&#x5EFA;&#x8BAE; Abl &#x901A;&#x8DEF;&#x4ECB;&#x5BFC;&#x7684;&#x591A;&#x4E2A;&#x56DE;&#x65CB;&#x53D7;&#x4F53;&#x4E0B;&#x6E38;&#x4FE1;&#x53F7;&#x3002;

&#x679C;&#x8747;&#x540C;&#x7CFB;&#x7269;&#x9178;&#x73AF;&#x5316;&#x9176;&#x76F8;&#x5173;&#x86CB;&#x767D;&#x7684;&#x534F;&#x540C;&#x63A7;&#x5236;&#x4E2D;&#x7EBF;&#x8F74;&#x7A81;&#x5BFB;&#x8DEF; Abl &#x916A;&#x6C28;&#x9178;&#x6FC0;&#x9176;&#x3002;

We demonstrate that Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. We find a robust and specific genetic interaction between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors.

A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding.

Nous d&eacute;montrons que capulet Drosophila (capt), un homologue de l&#39;ad&eacute;nylyl cyclase-associated protein qui lie et r&eacute;glemente l&#39;actine dans la levure, associe &agrave; Abl dans les cellules de la drosophile, sugg&eacute;rant une relation fonctionnelle in vivo. Nous trouvons une interaction g&eacute;n&eacute;tique entre le capitaine et l&#39;Abl robuste et pr&eacute;cise au point de choix de ligne m&eacute;diane o&ugrave; la croissance c&ocirc;ne r&eacute;pulsif fonctions pour restreindre la travers&eacute;e de l&#39;axone de la fente. Des interactions g&eacute;n&eacute;tiques entre le capitaine et fente prend en charge un mod&egrave;le o&ugrave; le Capt et Abl collaborent dans le cadre de la r&eacute;ponse de r&eacute;pulsive. Un soutien suppl&eacute;mentaire &agrave; ce mod&egrave;le est fourni par des interactions g&eacute;n&eacute;tiques affichant le capt et Abl avec plusieurs membres de la famille des r&eacute;cepteurs rond-point. Ces &eacute;tudes identifient les Capulet dans le cadre d&#39;une voie nouvelle reliant des signaux de guidage pour la r&eacute;gulation de la dynamique du cytosquelette et sugg&egrave;rent que la voie de l&#39;Abl est responsable des signaux en aval de plusieurs r&eacute;cepteurs rond-point.

Un homologue drosophile de prot&eacute;ines associ&eacute;es &agrave; l&#39;ad&eacute;nylcyclase collabore avec l&#39;Abl tyrosine kinase pour contr&ocirc;ler l&#39;orientation axon m&eacute;diane.

Ein Drosophila-Homolog von Cyclase-assoziierten Proteinen arbeitet mit der Abl-Tyrosinkinase zu Mittellinie Axon Wegfindung zu kontrollieren.

Dimostriamo che drosofila capulet (capt), un omologo dell&#39;adenilato ciclasi-associated protein che associa e regola actina in lievito, associa Abl nelle cellule di Drosophila, suggerendo un rapporto funzionale in vivo. Troviamo una robusta e specifica interazione genetica tra capt e Abl presso il punto di scelta della linea mediana dove la crescita del cono repellente Slit funzioni per limitare l&#39;incrocio assone. Interazioni genetiche tra capt e fessura supportano un modello dove Capt e Abl collaborare come parte della risposta repellente. Ulteriore supporto per questo modello &egrave; fornito dalle interazioni genetiche sia capt e Abl display con pi&ugrave; membri della famiglia del recettore rotonda. Questi studi identificano Capulet come parte di un percorso emergente che collega segnali guida al regolamento della dinamica del citoscheletro e suggeriscono che il pathway Abl da intermediario segnali a valle dei recettori pi&ugrave; rotonda.

Un omologo della drosofila di ciclasi-associated proteins collabora con l&#39;Abl tirosin-chinasi di controllare pathfinding assone della linea mediana.

&#x6211;&#x3005; &#x306F;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8; capulet (capt)&#x3001;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x304A;&#x3088;&#x3073;&#x9175;&#x6BCD;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x3068; in vivo &#x3067;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x95A2;&#x4FC2;&#x3092;&#x793A;&#x5506; Abl &#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x7D30;&#x80DE;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x307E;&#x3059;&#x3001;&#x30A2;&#x30C7;&#x30CB;&#x30EB;&#x9178;&#x30B7;&#x30AF;&#x30E9;&#x30FC;&#x30BC;&#x95A2;&#x9023;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x76F8;&#x540C;&#x7269;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x5805;&#x7262;&#x304B;&#x3064;&#x7279;&#x5B9A;&#x907A;&#x4F1D;&#x5B50;&#x3068;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528; capt Abl &#x6210;&#x9577;&#x5186;&#x9310;&#x64A5;&#x6B63;&#x4E2D;&#x7DDA;&#x306E;&#x9078;&#x629E;&#x70B9;&#x3067;&#x898B;&#x3064;&#x3051;&#x308B;&#x30B9;&#x30EA;&#x30C3;&#x30C8;&#x8EF8;&#x7D22;&#x6A2A;&#x65AD;&#x3092;&#x5236;&#x9650;&#x3059;&#x308B;&#x6A5F;&#x80FD;&#x3002;Capt &#x3068;&#x30B9;&#x30EA;&#x30C3;&#x30C8;&#x9593;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x76F8;&#x4E92;&#x4F5C;&#x7528; Capt &#x304A;&#x3088;&#x3073; Abl &#x306E;&#x64A5;&#x306E;&#x5FDC;&#x7B54;&#x306E;&#x4E00;&#x90E8;&#x3068;&#x3057;&#x3066;&#x5171;&#x540C;&#x4F5C;&#x696D;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x306E;&#x3055;&#x3089;&#x306A;&#x308B;&#x30B5;&#x30DD;&#x30FC;&#x30C8; capt &#x304A;&#x3088;&#x3073; Abl &#x8868;&#x793A;&#x30E9;&#x30A6;&#x30F3;&#x30C9; &#x30A2;&#x30D0;&#x30A6;&#x30C8;&#x4EA4;&#x5DEE;&#x70B9;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;&#x8907;&#x6570;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3068;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x3088;&#x3063;&#x3066;&#x63D0;&#x4F9B;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7814;&#x7A76;&#x306F; Capulet &#x7D30;&#x80DE;&#x9AA8;&#x683C;&#x306E;&#x52D5;&#x614B;&#x306E;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x8A98;&#x5C0E;&#x4FE1;&#x53F7;&#x3092;&#x30EA;&#x30F3;&#x30AF;&#x3001;&#x65B0;&#x8208;&#x306E;&#x7D4C;&#x8DEF;&#x306E;&#x4E00;&#x90E8;&#x3068;&#x3057;&#x3066;&#x8B58;&#x5225;&#x304A;&#x3088;&#x3073; Abl &#x7D4C;&#x8DEF;&#x304C;&#x8907;&#x6570;&#x30E9;&#x30A6;&#x30F3;&#x30C9; &#x30A2;&#x30D0;&#x30A6;&#x30C8;&#x4EA4;&#x5DEE;&#x70B9;&#x306E;&#x53D7;&#x5BB9;&#x5668;&#x306E;&#x4E0B;&#x6D41;&#x306E;&#x4FE1;&#x53F7;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x304A;&#x52E7;&#x3081;&#x3002;

&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x76F8;&#x540C;&#x7269;&#x30B7;&#x30AF;&#x30E9;&#x30FC;&#x30BC;&#x95A2;&#x9023;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x6B63;&#x4E2D;&#x7DDA;&#x306E;&#x8EF8;&#x7D22;&#x306E;&#x7D4C;&#x8DEF;&#x63A2;&#x7D22;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B; Abl &#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x3068;&#x9023;&#x643A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#xC6B0;&#xB9AC;&#xB294; &#xCD08;&#xD30C;&#xB9AC; capulet (&#xD568;&#xC7A5;) &#xBC14;&#xC778;&#xB529;&#xD569;&#xB2C8;&#xB2E4; &#xBC0F; &#xD6A8; &#xBAA8;&#xC5D0;&#xB294; &#xB9D0;&#xB77C; &#xADDC;&#xC81C;, Abl &#xCD08;&#xD30C;&#xB9AC; &#xC138;&#xD3EC;&#xC5D0; &#xC81C;&#xC548; &#xD558;&#xB294; in vivo &#xAE30;&#xB2A5; &#xAD00;&#xACC4;&#xB97C; &#xC5F0;&#xACB0; &#xD558;&#xB294; adenylyl cyclase &#xAD00;&#xB828; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xCCB4;&#xB97C; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xAC15;&#xB825; &#xD558; &#xACE0; &#xD2B9;&#xC815; &#xC720;&#xC804;&#xC790; &#xC0C1;&#xD638;&#xC791;&#xC6A9; &#xCEA1;&#xD2F4; Abl &#xC5B4;&#xB514; &#xC131;&#xC7A5; &#xCF58; &#xAD6C; &#xCDA9; &#xC81C; &#xC911;&#xAC04; &#xC120;&#xD0DD; &#xC9C0;&#xC810;&#xC5D0;&#xC11C; &#xCC3E;&#xC744; &#xC218; &#xADF8; &#xC5B4; &#xCD95; &#xC0AD; &#xD06C;&#xB85C; &#xC2F1;&#xC744; &#xC81C;&#xD55C; &#xD558;&#xB294; &#xAE30;&#xB2A5;. &#xCEA1;&#xD2F4;&#xACFC; &#xC2AC;&#xB9BF; &#xC0AC;&#xC774; &#xC720;&#xC804; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD568;&#xC7A5; &#xBC0F; Abl &#xD610;&#xC624; &#xAC10;&#xC744; &#xC751;&#xB2F5;&#xC758; &#xC77C;&#xBD80;&#xB85C; &#xACF5;&#xB3D9; &#xC791;&#xC5C5; &#xBAA8;&#xB378;&#xC744; &#xC9C0;&#xC6D0;. &#xC774; &#xBAA8;&#xB378;&#xC5D0; &#xB300; &#xD55C; &#xCD94;&#xAC00; &#xC9C0;&#xC6D0; &#xC720;&#xC804;&#xC790; &#xC0C1;&#xD638; &#xD568;&#xC7A5; &#xBC0F; Abl &#xD45C;&#xC2DC; &#xD558;&#xB294; &#xB85C;&#xD130;&#xB9AC; &#xC218;&#xC6A9; &#xCCB4; &#xAC00;&#xC871;&#xC758; &#xC5EC;&#xB7EC; &#xAD6C;&#xC131;&#xC6D0;&#xC5D0; &#xC758;&#xD574; &#xC81C;&#xACF5; &#xB429;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; cytoskeletal &#xC5ED;&#xD559;&#xC758; &#xADDC;&#xC81C; &#xC9C0;&#xCE68; &#xC2E0;&#xD638; &#xC5F0;&#xACB0; &#xC2E0;&#xD765; &#xACBD;&#xB85C;&#xC758; &#xC77C;&#xBD80;&#xB85C; Capulet &#xC2DD;&#xBCC4; &#xD558; &#xACE0; Abl &#xD1B5;&#xB85C; &#xD558;&#xB958; &#xC5EC;&#xB7EC; &#xB85C;&#xD130;&#xB9AC; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC2E0;&#xD638;&#xB97C; &#xC911;&#xC7AC;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

Drosophila &#xCCB4; cyclase &#xAD00;&#xB828; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC911;&#xAC04; &#xC120; &#xCD95; &#xC0AD; pathfinding &#xC81C;&#xC5B4; Abl tyrosine kinase&#xC640; &#xD611;&#xB825;.

Demostramos Drosophila capulet (capt), un hom&oacute;logo de la prote&iacute;na asociada a ciclasa del adenylyl que ata y regula la actina en la levadura, se asocia con Abl en c&eacute;lulas de Drosophila, sugiriendo una relaci&oacute;n funcional in vivo. Encontramos una interacci&oacute;n gen&eacute;tica robusta y espec&iacute;fica entre capt y Abl en el punto de elecci&oacute;n de la l&iacute;nea media donde el crecimiento cono repelente Raja funciones restringir el cruce del ax&oacute;n. Interacciones gen&eacute;ticas entre capt y Raja apoyan un modelo donde Capt y Abl colaboran como parte de la respuesta repelente. Ayuda adicional para este modelo es proporcionado por interacciones gen&eacute;ticas que muestran tanto capt y Abl con varios miembros de la familia del receptor de la rotonda. Estos estudios identifican Capulet como parte de un camino emergente que enlazan a las se&ntilde;ales de orientaci&oacute;n a la regulaci&oacute;n de la din&aacute;mica del citoesqueleto y sugieren que la v&iacute;a de la Abl media se&ntilde;ales aguas abajo de m&uacute;ltiples receptores rotonda.

Un hom&oacute;logo de Drosophila de prote&iacute;nas asociadas ciclasa colabora con la Abl tirosina quinasa controlar pathfinding axon de l&iacute;nea media.

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.

In Drosophila, the basic-helix-loop-helix protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, DIMM confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state.

Molecular organization of Drosophila neuroendocrine cells by Dimmed.

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.

Colony organization in the green alga Botryococcus braunii (Race B) is specified by a complex extracellular matrix.

Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, and Tail-up (Islet) to guide the stereotyped differentiation, connectivity, and function of a subset of neurons in Drosophila. The role of Hb9 in directing neuronal differentiation is well documented, but the lineage of Hb9(+) neurons is only partly characterized, its regulation is poorly understood, and most of the downstream genes through which it acts remain at large. Here, we complete the lineage tracing of all embryonic Hb9(+) neurons (to eight neuronal lineages) and provide evidence that hb9, lim3, and tail-up are coordinately regulated by a common set of upstream factors. Through the parallel use of micro-array gene expression profiling and the Dam-ID method, we searched for Hb9-regulated genes, uncovering transcription factors as the most over-represented class of genes regulated by Hb9 (and Nkx6) in the CNS. By a nearly ten-to-one ratio, Hb9 represses rather than activates transcription factors, highlighting transcriptional repression of other transcription factors as a core mechanism by which Hb9 governs neuronal determination. From the small set of genes activated by Hb9, we characterized the expression and function of two - fd59a/foxd, which encodes a transcription factor, and Nitric oxide synthase. Under standard lab conditions, both genes are dispensable for Drosophila development, but Nos appears to inhibit hyper-active behavior and fd59a appears to act in octopaminergic neurons to control egg-laying behavior. Together our data clarify the mechanisms through which Hb9 governs neuronal specification and differentiation and provide an initial characterization of the expression and function of Nos and fd59a in the Drosophila CNS.

Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of nitric oxide synthase and Fd59a/Fox-D.

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii.

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

Advanced methods of cellular purification are required to apply genome technology to the study of spermatogenesis. One approach, based on flow cytometry of murine testicular cells stained with Hoechst-33342 (Ho-FACS), has been extensively optimized and currently allows the isolation of 9 germ cell types. This staining technique is straightforward to implement, highly effective at purifying specific germ cell types and yields sufficient cell numbers for high throughput studies. Ho-FACS is a technique that does not require species-specific markers, but whose applicability to other species is largely unexplored. We hypothesized that, due to the similar cell physiology of spermatogenesis across mammals, Ho-FACS could be used to produce highly purified subpopulations of germ cells in mammals other than mouse. To test this hypothesis, we applied Ho-FACS to 4 mammalian species that are widely used in testis research - Rattus norvegicus, Cavia porcellus, Canis familiaris and Sus scrofa domesticus We successfully isolated 4 germ cell populations from these species with average purity of 79% for spermatocytes, and 90% for spermatids and 66% for spermatogonia. Additionally, we compare the performance of mechanical and chemical dissociation for each species, and propose an optimized gating strategy to better discriminate round and elongating spermatids in the mouse, which can potentially be applied to other species. Our work indicates that spermatogenesis may be uniquely accessible among mammalian developmental systems, as a single set of reagents may be sufficient to isolate germ cell populations from many different mammalian species, opening new avenues in the fields of development and male reproductive biology.

Jannette Rusch

Department of Genetics

Washington University School of Medicine

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Jannette Rusch

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics

Washington University School of Medicine

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Department of Genetics