manipulation-single-neural-stem-cells-neurons-brain-slices-using

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Max Planck Institute of Molecular Cell Biology and Genetics

&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x5927;&#x8111;&#x7684;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x671F;&#x95F4;&#x5DF2;&#x77E5;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x5728;&#x5FC3;&#x5BA4;&#x533A;&#x5E73;&#x5747;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x957F;&#x5EA6;&#x589E;&#x52A0;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x5BF9;&#x4E8E;&#x4EFB;&#x4F55;&#x7ED9;&#x5B9A;&#x7684;&#x533A;&#x57DF;&#x7684;&#x53D1;&#x5C55;&#x4E2D;&#x56FD;&#x5BB6;&#x7684;&#x76AE;&#x5C42;&#x548C;&#x795E;&#x7ECF;&#x53D1;&#x751F;&#x7684;&#x9636;&#x6BB5;&#xFF0C;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x7684;&#x957F;&#x5EA6;&#x88AB;&#x8BA4;&#x4E3A;&#x662F;&#x7C7B;&#x4F3C;&#x5728;&#x7956;&#x7EC6;&#x80DE; [&#x5373;&#xFF0C;&#x8FD9;&#x4E9B;&#x6B63;&#x5728;&#x8FDB;&#x884C;&#x7684; &#xFF08;&#x5BF9;&#x79F0;&#xFF09; &#x589E;&#x6B96;&#x5206;&#x88C2;&#x548C;&#x90A3;&#x4E9B;&#x7ECF;&#x5386; &#xFF08;&#x975E;&#x5BF9;&#x79F0;&#x6216;&#x5BF9;&#x79F0;&#xFF09; &#x795E;&#x7ECF;&#x5143;&#x4EA7;&#x751F;&#x5206;&#x6B67;] &#x4E24;&#x4E2A;&#x5E76;&#x5B58;&#x4E9A;&#x7FA4;&#x3002;&#x4F7F;&#x7528;&#x7D2F;&#x79EF;&#x6EB4;&#x8131;&#x6C27;&#x5C3F;&#x7519;&#x8D34;&#x6807; Tis21 &#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x6572;&#x5165;&#x5C0F;&#x9F20;&#x80DA;&#x80CE;&#xFF0C;&#x53EF;&#x4EE5;&#x533A;&#x5206;&#x8FD9;&#x4E9B;&#x4E24;&#x4E2A;&#x4E9A;&#x7FA4;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x4F53;&#x5185;&#xFF0C;&#x6211;&#x4EEC;&#x73B0;&#x5728;&#x663E;&#x793A;&#x53D1;&#x75C5;&#x4EE5;&#x53CA; telencephalic &#x795E;&#x7ECF;&#x518D;&#x751F;&#x7684;&#x9AD8;&#x7EA7;&#x7684;&#x9636;&#x6BB5;&#xFF0C;&#x5728;&#x7ECF;&#x5386;&#x795E;&#x7ECF;&#x5143;&#x4EA7;&#x751F;&#x5206;&#x6B67;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x7684;&#x7279;&#x70B9;&#x662F;&#x6BD4;&#x7956;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x5206;&#x88C2;&#x672F;&#x5927;&#x5927;&#x5EF6;&#x957F;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x3002;&#x53E6;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x6700;&#x8FD1;&#x4E3A;&#x7279;&#x5F81;&#x7684;&#x795E;&#x7ECF;&#x524D;&#x4F53;&#x7EC6;&#x80DE;&#x5728;&#x57FA;&#x5E95;&#x4FA7;&#x8111;&#x5BA4;&#x533A;&#xFF0C;&#x5BA4;&#x7BA1;&#x819C;&#x4E0B;&#x533A;&#x5212;&#x5206;&#x6709;&#x6BD4;&#x90A3;&#x4E9B;&#x5728;&#x5FC3;&#x5BA4;&#x8868;&#x9762;&#x5212;&#x5206;&#x957F; G(2) &#x9636;&#x6BB5;&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x90FD;&#x7B26;&#x5408;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x5EF6;&#x957F;&#x56E0;&#x679C;&#x5173;&#x7CFB;&#x53EF;&#x4EE5;&#x4FC3;&#x8FDB;&#x795E;&#x7ECF;&#x7956;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x4ECE;&#x5207;&#x6362;&#x5230;&#x795E;&#x7ECF;&#x5143;&#x4EA7;&#x751F;&#x5206;&#x6B67;&#x548C; 5 &#x6708;&#x7684;&#x5047;&#x8BF4; &#xFF08;&#x5361;&#x83B1;&#x52A0;&#x91CC;&#x548C; Huttner&#xFF0C;2003&#x5E74;&#xFF09; &#x4E00;&#x822C;&#x5177;&#x6709;&#x91CD;&#x8981;&#x5F71;&#x54CD;&#x7684;&#x6210;&#x4F53;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x6269;&#x5F20;&#x3002;

&#x9009;&#x62E9;&#x6027;&#x5EF6;&#x957F;&#x5728;&#x795E;&#x7ECF;&#x6E90;&#x6027;&#x795E;&#x7ECF;&#x7956;&#x7EC6;&#x80DE;&#x4E9A;&#x7FA4;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x671F;&#x95F4;&#x9F20;&#x6807;&#x5927;&#x8111;&#x53D1;&#x80B2;&#x3002;

During embryonic development of the mammalian brain, the average cell-cycle length of progenitor cells in the ventricular zone is known to increase. However, for any given region of the developing cortex and stage of neurogenesis, the length of the cell cycle is thought to be similar in the two coexisting subpopulations of progenitors [i.e., those undergoing (symmetric) proliferative divisions and those undergoing (either asymmetric or symmetric) neuron-generating divisions]. Using cumulative bromodeoxyuridine labeling of Tis21-green fluorescent protein knock-in mouse embryos, in which these two subpopulations of progenitors can be distinguished in vivo, we now show that at the onset as well as advanced stages of telencephalic neurogenesis, progenitors undergoing neuron-generating divisions are characterized by a significantly longer cell cycle than progenitors undergoing proliferative divisions. In addition, we find that the recently characterized neuronal progenitors dividing at the basal side of the ventricular zone and in the subventricular zone have a longer G(2) phase than those dividing at the ventricular surface. These findings are consistent with the hypothesis (Calegari and Huttner, 2003) that cell-cycle lengthening can causally contribute to neural progenitors switching from proliferative to neuron-generating divisions and may have important implications for the expansion of somatic stem cells in general.

Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development.

Durant le d&eacute;veloppement embryonnaire du cerveau chez les mammif&egrave;res, la longueur moyenne du cycle cellulaire des cellules prog&eacute;nitrices dans la zone ventriculaire est connue pour augmenter. Toutefois, pour toutes les r&eacute;gions du cortex en d&eacute;veloppement et les &eacute;tapes de la neurogen&egrave;se, la longueur du cycle cellulaire est consid&eacute;r&eacute;e comme semblable dans les deux sous-populations coexistantes de prog&eacute;niteurs [c&#39;est-&agrave;-dire, ces divisions prolif&eacute;ratives (sym&eacute;triques) en cours et ceux qui subissent des divisions de g&eacute;n&eacute;ratrices de neurone (sym&eacute;triques ou asym&eacute;triques)]. L&#39;utilisation cumulative bromod&eacute;oxyuridine &eacute;tiquetage des embryons de souris knock-in Tis21-vert fluorescent protein, dans lequel ces deux sous-populations de cellules souches peuvent &ecirc;tre distingu&eacute;es in vivo, nous montrons maintenant qu&#39;&agrave; l&#39;apparition ainsi que les stades avanc&eacute;s de la neurogen&egrave;se t&eacute;lenc&eacute;phaliques, prog&eacute;niteurs subissant des divisions de g&eacute;n&eacute;ratrices de neurone sont caract&eacute;risent par un cycle cellulaire beaucoup plus longs que les prog&eacute;niteurs subissant des divisions prolif&eacute;ratives. En outre, nous constatons que les prog&eacute;niteurs neurones r&eacute;cemment caract&eacute;ris&eacute;es divisant &agrave; la partie basale de la zone ventriculaire et dans la zone sous-ventriculaire ont une phase prolong&eacute;e de montrons que ceux divisant la surface ventriculaire. Ces r&eacute;sultats concordent avec l&#39;hypoth&egrave;se que l&#39;allongement du cycle cellulaire peut causalement contribuer &agrave; prog&eacute;niteurs neuraux passant prolif&eacute;rative aux g&eacute;n&eacute;ratrices de neurone divisions et mai (Calegari et Huttner, 2003) ont des implications importantes pour l&#39;expansion des cellules souches somatiques en g&eacute;n&eacute;ral.

S&eacute;lectif allongement du cycle cellulaire dans la sous-population neurog&egrave;ne de cellules prog&eacute;nitrices neurales au cours du d&eacute;veloppement de cerveau de souris.

W&auml;hrend der Embryonalentwicklung der das Gehirn von S&auml;ugetieren ist die durchschnittliche Zellzyklus von Progenitorzellen bei der ventrikul&auml;ren Zone bekannt, zu erh&ouml;hen. Allerdings ist f&uuml;r jeden gegebenen Region der entwickelnden Rinde und B&uuml;hne der Neurogenese, die L&auml;nge des Zellzyklus dachte, in der zwei nebeneinander existierende Subpopulationen Vorreiter [d.h. diese Gegenstand (symmetrischen) proliferativen Trennlinien und diejenigen unterzogen (asymmetrische oder symmetrische) Neuron schaffende Divisionen] &auml;hnlich sein. Mit kumulativen Bromdesoxyuridin Kennzeichnung Tis21-gr&uuml;n fluoreszierendes Protein Knock-in-Maus Embryonen, in denen diese zwei Subpopulationen von Vorl&auml;uferzellen in vivo unterschieden werden k&ouml;nnen zeigen wir jetzt, dass der Beginn als auch fortgeschrittenen Stadien der Teleenzephalischer Neurogenese, Vorfahre in Neuron schaffende Divisionen durch eine erheblich l&auml;ngere Zellzyklus als Vorfahre proliferative Abteilungen unterzogen gekennzeichnet sind. Au&szlig;erdem finden wir, dass die vor kurzem gekennzeichneten neuronalen Vorl&auml;uferzellen, die Division auf der basalen Seite der ventrikul&auml;ren Zone und in der subventrikul&auml;re Zone eine l&auml;ngere Fassung-Phase als die Division an der ventrikul&auml;ren Oberfl&auml;che haben. Diese Ergebnisse stehen im Einklang mit der Hypothese (Calegari und Huttner, 2003), dass die Zellzyklus Verl&auml;ngerung kausal zu neuronalen Vorl&auml;uferzellen Wechsel von proliferative Neuron erzeugen Divisionen und Mai beitragen kann erhebliche Auswirkungen f&uuml;r den Ausbau der somatische Stammzellen im Allgemeinen haben.

Selektive Verl&auml;ngerung des Zellzyklus in die neurogene Teilgesamtheit von neuronalen Vorl&auml;uferzellen w&auml;hrend Maus Gehirnentwicklung.

Durante lo sviluppo embrionale del cervello dei mammiferi, la lunghezza media di ciclo cellulare delle cellule progenitrici nella zona ventricolare &egrave; noto per aumentare. Tuttavia, per ogni determinata regione della corteccia in via di sviluppo e fase di neurogenesi, la lunghezza del ciclo cellulare &egrave; pensata per essere simile in due sottopopolazioni coesistenti di progenitori [cio&egrave;, quelle divisioni proliferative (simmetriche) in corso e quelli in fase di divisioni neurone-generazione (sia simmetriche o asimmetriche)]. Utilizzando bromodeoxyuridine cumulativa di etichettatura degli embrioni di topo knock-in proteina fluorescente verde-Tis21, in cui questi due sottopopolazioni di progenitori possono essere distinto in vivo, mostriamo ora che l&#39;insorgenza, nonch&eacute; fasi avanzate della neurogenesi telencefalici, progenitori in fase di generazione di neurone divisioni sono caratterizzati da un ciclo cellulare significativamente pi&ugrave; lungo rispetto a progenitori in fase proliferative divisioni. Inoltre, troviamo che i progenitori neuronali recentemente caratterizzati dividendo sul lato basale della zona ventricolare e nella zona subventricolare hanno una fase 05 pi&ugrave; lunga rispetto a quelli dividendo la superficie ventricolare. Questi risultati sono coerenti con l&#39;ipotesi (Calegari e Huttner, 2003) che allungamento del ciclo cellulare causale pu&ograve; contribuire a progenitori neurali di commutazione da proliferativa al neurone-generando divisioni e possono avere importanti implicazioni per l&#39;espansione di cellule staminali somatiche in generale.

Allungamento selettivo del ciclo cellulare nella neurogena sottopopolazione di cellule progenitrici neurali durante lo sviluppo del cervello del mouse.

&#x54FA;&#x4E73;&#x985E;&#x306E;&#x8133;&#x306E;&#x840C;&#x82BD;&#x671F;&#x306E;&#x958B;&#x767A;&#x4E2D;&#x306F;&#x3001;&#x5FC3;&#x5BA4;&#x306E;&#x30BE;&#x30FC;&#x30F3;&#x306B;&#x304A;&#x3051;&#x308B;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x306E;&#x5E73;&#x5747;&#x9577;&#x3055;&#x3092;&#x5897;&#x3084;&#x3059;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x4EFB;&#x610F;&#x306E;&#x5927;&#x8133;&#x76AE;&#x8CEA;&#x306E;&#x4E0E;&#x3048;&#x3089;&#x308C;&#x305F;&#x9818;&#x57DF;&#x3068;&#x795E;&#x7D4C;&#x65B0;&#x751F;&#x306E;&#x6BB5;&#x968E;&#x306B;&#x3064;&#x3044;&#x3066;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x306E;&#x9577;&#x3055;&#x524D;&#x99C6;&#x7D30;&#x80DE; [&#x3059;&#x306A;&#x308F;&#x3061;&#x3001;&#x305D;&#x308C;&#x3089;&#x53D7;&#x3051; (&#x5BFE;&#x79F0;) &#x5897;&#x6B96;&#x90E8;&#x9580;&#x3068;&#x305D;&#x308C;&#x3089; (&#x975E;&#x5BFE;&#x79F0;&#x307E;&#x305F;&#x306F;&#x5BFE;&#x79F0;) &#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x90E8;&#x9580;&#x3092;&#x53D7;&#x3051;&#x3066;&#x3044;&#x308B;] &#x306E; 2 &#x3064;&#x306E;&#x5171;&#x5B58;&#x3059;&#x308B;&#x96C6;&#x56E3;&#x3067;&#x4F3C;&#x3066;&#x3044;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x307E;&#x3059;&#x3002;&#x7D2F;&#x7A4D;&#x30D6;&#x30ED;&#x30E2;&#x30C7;&#x30AA;&#x30AD;&#x30B7;&#x30A6;&#x30EA;&#x30B8;&#x30F3;&#x3067;&#x3053;&#x308C;&#x3089; 2 &#x3064;&#x306E;&#x30B5;&#x30D6;&#x30DD;&#x30D4;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E; in vivo &#x3067;&#x533A;&#x5225;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059; Tis21 &#x7DD1;&#x8272;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30CE;&#x30C3;&#x30AF;&#x30A4;&#x30F3; &#x30DE;&#x30A6;&#x30B9;&#x80DA;&#x306E;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x4ECA;&#x3001;&#x767A;&#x75C7;&#x3068;&#x540C;&#x69D8;&#x306B;&#x80CE;&#x751F;&#x795E;&#x7D4C;&#x65B0;&#x751F;&#x306E;&#x9032;&#x884C;&#x3057;&#x305F;&#x6BB5;&#x968E;&#x3001;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x90E8;&#x9580;&#x3092;&#x53D7;&#x3051;&#x3066;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x90E8;&#x9580;&#x3092;&#x53D7;&#x3051;&#x3066;&#x3088;&#x308A;&#x5927;&#x5E45;&#x306B;&#x9577;&#x304F;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x306B;&#x3088;&#x3063;&#x3066;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x308B;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x5206;&#x5272;&#x57FA;&#x5E95;&#x5074;&#x306E;&#x5FC3;&#x5BA4;&#x306E;&#x30BE;&#x30FC;&#x30F3;&#x3068;&#x8133;&#x5BA4;&#x4E0B;&#x5E2F;&#x3067;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x308B;&#x3001;&#x6700;&#x8FD1;&#x795E;&#x7D4C;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x304C;&#x9577;&#x304F; G(2) &#x76F8;&#x3088;&#x308A;&#x3082;&#x5FC3;&#x5BA4;&#x306E;&#x8868;&#x9762;&#x3067;&#x5206;&#x5272;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x898B;&#x3064;&#x3051;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x6240;&#x898B;&#x306F;&#x795E;&#x7D4C;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x5897;&#x6B96;&#x304B;&#x3089;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x90E8;&#x9580;&#x3068; 5 &#x6708;&#x306B;&#x30B9;&#x30A4;&#x30C3;&#x30C1;&#x30F3;&#x30B0;&#x3059;&#x308B;&#x306B;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x3092;&#x5EF6;&#x9577;&#x56E0;&#x679C;&#x7684;&#x8CA2;&#x732E;&#x3067;&#x304D;&#x308B;&#x3068;&#x4EEE;&#x8AAC; (Calegari &#x3068; Huttner&#x3001;2003&#x5E74;&#xFF09; &#x3068;&#x4E00;&#x81F4;&#x3059;&#x308B;&#x4E00;&#x822C;&#x306B;&#x4F53;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x62E1;&#x5927;&#x306E;&#x91CD;&#x8981;&#x306A;&#x542B;&#x610F;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x9078;&#x629E;&#x7684;&#x795E;&#x7D4C;&#x56E0;&#x6027;&#x4E9C;&#x795E;&#x7D4C;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x8133;&#x306E;&#x767A;&#x9054;&#x4E2D;&#x306B;&#x5EF6;&#x9577;&#x3002;

&#xD3EC;&#xC720;&#xB958; &#xB450;&#xB1CC;&#xC758; &#xBC30;&#xC544; &#xBC1C;&#xB2EC; &#xB3D9;&#xC548; &#xC2EC; &#xC2E4; &#xC601;&#xC5ED;&#xC5D0;&#xC11C; &#xC870;&#xC0C1; &#xC138;&#xD3EC;&#xC758; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xD3C9;&#xADE0; &#xAE38;&#xC774; &#xC99D;&#xAC00; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xAC1C;&#xBC1C; &#xD53C; &#xC9C8;&#xC758; &#xD2B9;&#xC815;&#xB41C; &#xC601;&#xC5ED; &#xC5B4;&#xB5A4; &#xC131;&#xCCB4;&#xC758; &#xB2E8;&#xACC4;&#xC5D0; &#xB300;&#xD574;&#xC11C; &#xC138;&#xD3EC; &#xC8FC;&#xAE30;&#xC758; &#xAE38;&#xC774; &#xBE44;&#xC2B7;&#xD55C; progenitors [&#xC989;, &#xADF8; &#xC9C4;&#xD589; (&#xB300;&#xCE6D;) &#xC99D;&#xC2DD; &#xBD80;&#xBB38; &#xBC0F; (&#xBE44;&#xB300;&#xCE6D; &#xB610;&#xB294; &#xB300;&#xCE6D;) &#xC2E0;&#xACBD; &#xC0DD;&#xC131; &#xBD84;&#xC5F4;&#xC744; &#xACAA;&#xACE0; &#xADF8;]&#xC758; &#xACF5;&#xC874; &#xB450; &#xC9D1;&#xB2E8;&#xAC04;&#xC5D0; &#xB420; &#xC0DD;&#xAC01; &#xC774;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC9C0;&#xAE08;&#xC5D0; &#xBC1C;&#xBCD1;&#xC73C;&#xB85C; telencephalic &#xC131;&#xCCB4;&#xC758; &#xACE0;&#xAE09; &#xB2E8;&#xACC4;, progenitors &#xC2E0;&#xACBD; &#xC0DD;&#xC131; &#xBD84;&#xC5F4;&#xC744; &#xACAA;&#xACE0; &#xC99D;&#xC2DD; &#xBD84;&#xC5F4;&#xC744; &#xACAA;&#xACE0; progenitors &#xBCF4;&#xB2E4; &#xD6E8;&#xC52C; &#xB354; &#xAE34; &#xC138;&#xD3EC; &#xC8FC;&#xAE30;&#xC5D0; &#xC758;&#xD574; &#xD2B9;&#xC9D5;&#xC740; &#xD45C;&#xC2DC; &#xB204;&#xC801; bromodeoxyuridine&#xB294; progenitors&#xC758; &#xB450; &#xC9D1;&#xB2E8;&#xAC04;&#xC774; &#xAD6C;&#xBD84;&#xD560; &#xC218; vivo&#xC5D0;&#xC11C; Tis21 &#xB179;&#xC0C9; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8; &#xB178;&#xD06C; &#xB9C8;&#xC6B0;&#xC2A4; &#xBC30;&#xC544;&#xC758; &#xB77C;&#xBCA8;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xCD5C;&#xADFC;&#xC5D0; &#xD2B9;&#xC9D5;&#xC774; &#xC2E0;&#xACBD; progenitors &#xC2EC; &#xC2E4; &#xC601;&#xC5ED; &#xBC0F; &#x5E2F; &#xAE30;&#xC800; &#xBA74;&#xC744; &#xB098;&#xB204;&#xC5B4;&#xAC00;&#xC9C0;&#xACE0; &#xC2EC; &#xC2E4; &#xD45C;&#xBA74;&#xC5D0; &#xB098;&#xB204;&#xB294; &#xAC83; &#xBCF4;&#xB2E4; &#xB354; &#xAE34; G(2) &#xB2E8;&#xACC4; &#xCC3E;&#xC744;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xAE38;&#xC5B4; &#xC2E0;&#xACBD; progenitors &#xC99D;&#xC2DD;&#xC5D0;&#xC11C; &#xC2E0;&#xACBD; &#xC0DD;&#xC131; &#xBD80;&#xC11C;&#xC640; 5 &#xC6D4;&#xC5D0; &#xC804;&#xD658;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; causally &#xC218; &#xAC00;&#xC124; (Calegari &#xBC0F; Huttner, 2003)&#xC640; &#xC77C;&#xCE58; &#xD558;&#xB294; &#xC77C;&#xBC18; &#xCCB4;&#xC138;&#xD3EC; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xD655;&#xC7A5;&#xC744; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xC758;&#xBBF8;&#xB97C;&#xAC00;&#xC9C0;&#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC120;&#xD0DD;&#xC801; &#xAE38;&#xC5B4; neurogenic subpopulation &#xC2E0;&#xACBD; &#xC870;&#xC0C1; &#xC138;&#xD3EC;&#xC758; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xB9C8;&#xC6B0;&#xC2A4; &#xB450;&#xB1CC; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548;.

Durante el desarrollo embrionario del cerebro mam&iacute;fero, la longitud de ciclo celular promedio de c&eacute;lulas progenitoras en la zona ventricular se conoce para aumentar. Sin embargo, para cualquier regi&oacute;n de la corteza en desarrollo y etapa de la neurog&eacute;nesis, la longitud del ciclo celular es probablemente similar en las dos subpoblaciones coexistentes de progenitores [es decir, esas divisiones proliferativas (sim&eacute;tricas) que experimenta y los sometidos a divisiones generadoras de neurona (sim&eacute;tricas o asim&eacute;tricas)]. Utilizando bromodeoxiuridina acumulativa etiquetado de embriones de rat&oacute;n knock-in de prote&iacute;na fluorescente verde Tis21, en el cual se pueden distinguir estas dos subpoblaciones de progenitores in vivo, ahora mostramos que en el inicio as&iacute; como las etapas avanzadas de la neurog&eacute;nesis telenc&eacute;falo, progenitores sometidos a neurona-generando divisiones se caracterizan por un ciclo celular significativamente mayor que los progenitores sometidos a divisiones proliferativas. Adem&aacute;s, encontramos que el trasplante de precursores neuronales recientemente caracterizado dividiendo en la parte basal de la zona ventricular y en la zona subventricular tiene una fase m&aacute;s larga de la f&oacute;rmula que los divide en la superficie ventricular. Estos resultados son consistentes con la hip&oacute;tesis (Calegari y Huttner, 2003) que alargar el ciclo celular causal puede contribuir a progenitores neuronales conmutaci&oacute;n de proliferativa a neurona-generar divisiones y mayo tienen implicaciones importantes para la expansi&oacute;n de c&eacute;lulas som&aacute;ticas en general.

Alargamiento selectivo del ciclo celular en la subpoblaci&oacute;n neurog&eacute;nica de c&eacute;lulas progenitoras neurales durante el desarrollo del cerebro del rat&oacute;n.

Basal (intermediate) progenitors are the major source of neurons in the mammalian neocortex. The molecular machinery governing basal progenitor biogenesis is unknown. Here, we show that the zinc-finger transcription factor Insm1 (insulinoma-associated 1) is expressed specifically in progenitors undergoing neurogenic divisions, has a panneurogenic role throughout the brain, and promotes basal progenitor formation in the neocortex. Mouse embryos lacking Insm1 contained half the number of basal progenitors and showed a marked reduction in cortical plate radial thickness. Forced premature expression of Insm1 in neuroepithelial cells resulted in their mitosis occurring at the basal (rather than apical) side of the ventricular zone and induced expression of the basal progenitor marker Tbr2. Remarkably, these cells remained negative for Tis21, a marker of neurogenic progenitors, and did not generate neurons but underwent self-amplification. Our data imply that Insm1 is involved in the generation and expansion of basal progenitors, a hallmark of neocortex evolution.

Insulinoma-associated 1 has a panneurogenic role and promotes the generation and expansion of basal progenitors in the developing mouse neocortex.

Neurogenesis during the development of the mammalian cerebral cortex involves a switch of neural stem and progenitor cells from proliferation to differentiation. To explore the possible role of microRNAs (miRNAs) in this process, we conditionally ablated Dicer in the developing mouse neocortex using Emx1-Cre, which is specifically expressed in the dorsal telencephalon as early as embryonic day (E) 9.5. Dicer ablation in neuroepithelial cells, which are the primary neural stem and progenitor cells, and in the neurons derived from them, was evident from E10.5 onwards, as ascertained by the depletion of the normally abundant miRNAs miR-9 and miR-124. Dicer ablation resulted in massive hypotrophy of the postnatal cortex and death of the mice shortly after weaning. Analysis of the cytoarchitecture of the Dicer-ablated cortex revealed a marked reduction in radial thickness starting at E13.5, and defective cortical layering postnatally. Whereas the former was due to neuronal apoptosis starting at E12.5, which was the earliest detectable phenotype, the latter reflected dramatic impairment of neuronal differentiation. Remarkably, the primary target cells of Dicer ablation, the neuroepithelial cells, and the neurogenic progenitors derived from them, were unaffected by miRNA depletion with regard to cell cycle progression, cell division, differentiation and viability during the early stage of neurogenesis, and only underwent apoptosis starting at E14.5. Our results support the emerging concept that progenitors are less dependent on miRNAs than their differentiated progeny, and raise interesting perspectives as to the expansion of somatic stem cells.

miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex.

&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x7684;&#x5927;&#x8111;&#x76AE;&#x8D28;&#x53D1;&#x80B2;&#x8FC7;&#x7A0B;&#x5DF2;&#x77E5;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x7684; G1 &#x671F;&#x5EF6;&#x957F;&#xFF0C;&#x4F46;&#x4E00;&#x76F4;&#x4E0D;&#x6E05;&#x695A;&#x5176;&#x4E2D;&#x795E;&#x7ECF;&#x5E72;&#x548C;&#x53D7;&#x5F71;&#x54CD;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x3002;&#x5728;&#x672C;&#x767D;&#x76AE;&#x4E66;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86;&#x4E00;&#x79CD;&#x65B0;&#x7684;&#x65B9;&#x6CD5;&#x6765;&#x786E;&#x5B9A;&#x7279;&#x5B9A;&#x7C7B;&#x795E;&#x7ECF;&#x5E72;&#x548C;&#x7956;&#x7EC6;&#x80DE;&#xFF0C;&#x7531;&#x5206;&#x5B50;&#x6807;&#x8BB0;&#xFF0C;&#x800C;&#x4E0D;&#x662F;&#x4F4D;&#x7F6E;&#x6807;&#x8BC6;&#x4E2D;&#x7684;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x53C2;&#x6570;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0; G1 &#x5EF6;&#x957F;&#x4ECE;&#x5E72;&#x7EC6;&#x80DE;&#x6837;&#x5FC3;&#x5C16;&#x7956;&#x7EC6;&#x80DE;&#x8FC7;&#x6E21;&#x5230;&#x547D;&#x8FD0;&#x9650;&#x5236;&#x57FA;&#x5E95; &#xFF08;&#x4E2D;&#x95F4;&#xFF09; &#x7956;&#x7EC6;&#x80DE;&#x4E0E;&#x76F8;&#x5173;&#x8054;&#x3002;&#x610F;&#x5916;&#x7684;&#x662F;&#xFF0C;&#x6269;&#x5927;&#x5FC3;&#x5C16;&#x548C;&#x57FA;&#x5E95;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x8868;&#x73B0;&#x51FA;&#x66F4;&#x5927;&#x5927;&#x52A0;&#x957F; S-&#x671F;&#x6BD4;&#x81F4;&#x529B;&#x4E8E;&#x795E;&#x7ECF;&#x5143;&#x751F;&#x4EA7;&#x7684;&#x6839;&#x5C16;&#x548C;&#x57FA;&#x5E95;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x3002;&#x6269;&#x5927;&#x4E0E;&#x81F4;&#x529B;&#x4E8E;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x6BD4;&#x8F83;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x5206;&#x6790;&#x63ED;&#x793A;&#x4E86;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x8C03;&#x63A7;&#x3001; DNA &#x590D;&#x5236;&#x548C;&#x4FEE;&#x590D;&#x548C;&#x67D3;&#x8272;&#x8D28;&#x91CD;&#x5851;&#x7684;&#x5173;&#x952E;&#x56E0;&#x7D20;&#x7684;&#x53D8;&#x5316;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x6269;&#x5927;&#x795E;&#x7ECF;&#x5E72;&#x7956;&#x7EC6;&#x80DE;&#x6295;&#x8D44;&#x66F4;&#x591A;&#x7684;&#x65F6;&#x95F4;&#x5728; S &#x9636;&#x6BB5;&#x8D28;&#x91CF;&#x63A7;&#x5236;&#x4E2D;&#x590D;&#x5236; DNA &#x6BD4;&#x90A3;&#x4E9B;&#x81F4;&#x529B;&#x4E8E;&#x795E;&#x7ECF;&#x5143;&#x751F;&#x4EA7;&#x3002;

&#x795E;&#x7ECF;&#x5E72;&#x548C;&#x7956;&#x7EC6;&#x80DE;&#x5BF9;&#x795E;&#x7ECF;&#x5143;&#x751F;&#x4EA7;&#x627F;&#x8BFA;&#x7F29;&#x77ED; S &#x9636;&#x6BB5;&#x3002;

During mammalian cerebral cortex development, the G1-phase of the cell cycle is known to lengthen, but it has been unclear which neural stem and progenitor cells are affected. In this paper, we develop a novel approach to determine cell-cycle parameters in specific classes of neural stem and progenitor cells, identified by molecular markers rather than location. We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors. Unexpectedly, expanding apical and basal progenitors exhibit a substantially longer S-phase than apical and basal progenitors committed to neuron production. Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling. Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

Neural stem and progenitor cells shorten S-phase on commitment to neuron production.

Au cours du d&eacute;veloppement chez les mammif&egrave;res le cortex c&eacute;r&eacute;bral, la phase G1 du cycle cellulaire est connue &agrave; s&#39;allonger, mais on sait pas qui neuronal de la tige et les cellules prog&eacute;nitrices sont affect&eacute;es. Dans cet article, nous d&eacute;veloppons une nouvelle approche pour d&eacute;terminer les param&egrave;tres du cycle cellulaire dans des classes sp&eacute;cifiques de cellules souches et prog&eacute;nitrices neurales, identifi&eacute;s par marqueurs mol&eacute;culaires plut&ocirc;t qu&#39;emplacement. Nous avons trouv&eacute; que le G1 allongement &eacute;tait li&eacute;e &agrave; la transition de prog&eacute;niteurs apicales de cellules souches ressemblant au sort restreints basales prog&eacute;niteurs (interm&eacute;diaires). Contre toute attente, en pleine expansion des prog&eacute;niteurs apicales et basales pr&eacute;sentent une phase S nettement plus long que les prog&eacute;niteurs basales et apicales, commis &agrave; la production de neurones. Comparative genome-wide gene expression analysis of expansion par rapport &agrave; des cellules prog&eacute;nitrices engag&eacute;s ont r&eacute;v&eacute;l&eacute; des changements dans les facteurs cl&eacute;s de la r&eacute;gulation du cycle cellulaire, la r&eacute;plication de l&#39;ADN et r&eacute;paration et remodelage de la chromatine. Nos r&eacute;sultats sugg&egrave;rent qu&#39;expansion neuronal de la tige et cellules prog&eacute;nitrices investissent plus de temps au cours de la phase S en contr&ocirc;le de la qualit&eacute; de l&#39;ADN r&eacute;pliqu&eacute; que ceux commis &agrave; la production de neurones.

Les cellules souches et prog&eacute;nitrices neurales raccourcissent la phase S sur l&#39;engagement &agrave; la production de neurones.

W&auml;hrend der Entwicklung der S&auml;ugetier-Hirnrinde die G1-Phase des Zellzyklus ist bekannt, dass zu verl&auml;ngern, aber es war unklar welche neuronalen Stammzellen und Vorl&auml;uferzellen sind betroffen. In diesem Papier entwickeln wir einen neuen Ansatz, um zu bestimmen, Zellzyklus Parameter in bestimmten Klassen von Stamm- und Vorl&auml;uferzellen Nervenzellen, durch molekulare Marker anstatt Lage identifiziert. Wir fanden, dass G1 Verl&auml;ngerung der &Uuml;bergang von Stammzell-wie apikale Vorl&auml;uferzellen zu Schicksal eingeschr&auml;nkten basal (Mittelstufe) Vorl&auml;uferzellen zugeordnet. Unerwartet, expandierende apikale und basale Vorl&auml;uferzellen weisen eine wesentlich l&auml;ngere S-Phase als apikale und basale Vorl&auml;uferzellen Neuron Produktion verpflichtet. Vergleichende genomweiten Genexpressionsanalyse der Erweiterung gegen&uuml;ber verpflichtet Vorl&auml;uferzellen offenbarten Schl&uuml;sselfaktoren der Zellzykluskontrolle-, DNA-Replikation und Reparatur und Umbau von Chromatin. Unsere Ergebnisse weisen erweitern neuronalen Stammzellen und Vorl&auml;uferzellen investieren mehr Zeit in S-Phase in der Qualit&auml;tskontrolle von replizierten DNA als die Neuron-Produktion verpflichtet.

Stamm- und Vorl&auml;uferzellen Nervenzellen verk&uuml;rzen die S-Phase Bekenntnis zu Neuron-Produktion.

Durante lo sviluppo della corteccia cerebrale dei mammiferi, la fase G1 del ciclo cellulare &egrave; noto per allungare, ma &egrave; stato chiaro che neurali staminali e le cellule progenitrici sono colpite. In questa carta, sviluppiamo un nuovo approccio per determinare i parametri del ciclo cellulare nelle specifiche classi di cellule staminali e progenitrici neurali, identificate da marcatori molecolari piuttosto che posizione. Abbiamo trovato che allungamento G1 &egrave; stata associata con la transizione da progenitori apicale delle cellule staminali-come a limitato destino progenitori basale (intermedi). Inaspettatamente, espansione progenitori apicale e basale esibiscono una fase S sostanzialmente pi&ugrave; lunga di progenitori apicale e basale, impegnata nella produzione di neurone. Analisi di espressione comparativa genome-wide gene di espansione contro le cellule progenitrici impegnata ha rivelato cambiamenti nei fattori chiave della regolazione del ciclo cellulare, la replicazione del DNA e riparazione e rimodellamento della cromatina. I nostri risultati suggeriscono che espandendo neurali staminali e cellule progenitrici investono pi&ugrave; tempo durante la fase S in controllo di qualit&agrave; della replicato del DNA di coloro che si dedicano alla produzione del neurone.

Cellule staminali e progenitrici neurali accorciano la fase S sull&#39;impegno per la produzione del neurone.

&#x54FA;&#x4E73;&#x985E;&#x5927;&#x8133;&#x76AE;&#x8CEA;&#x306E;&#x958B;&#x767A;&#x4E2D;&#x306B;&#x7D30;&#x80DE;&#x5468;&#x671F; G1 &#x671F;&#x3092;&#x9577;&#x304F;&#x306B;&#x306F;&#x3001;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x305D;&#x308C;&#x306F;&#x795E;&#x7D4C;&#x306E;&#x5E79;&#x30AF;&#x30EA;&#x30A2;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x3092;&#x53D7;&#x3051;&#x307E;&#x3059;&#x3002;&#x672C;&#x7A3F;&#x3067;&#x306F;&#x3001;&#x7279;&#x5B9A;&#x30AF;&#x30E9;&#x30B9;&#x306E;&#x5206;&#x5B50;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3067;&#x306F;&#x306A;&#x304F;&#x3001;&#x5834;&#x6240;&#x306B;&#x3088;&#x3063;&#x3066;&#x8B58;&#x5225;&#x3055;&#x308C;&#x308B;&#x795E;&#x7D4C;&#x306E;&#x5E79;&#x30FB;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x30D1;&#x30E9;&#x30E1;&#x30FC;&#x30BF;&#x30FC;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x65AC;&#x65B0;&#x306A;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3059;&#x3002;G1 &#x3092;&#x5EF6;&#x9577;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x3088;&#x3046;&#x306A;&#x6839;&#x5C16;&#x304B;&#x3089;&#x904B;&#x547D;&#x5236;&#x9650;&#x57FA;&#x5E95; (&#x4E2D;&#x9593;) &#x524D;&#x99C6;&#x7D30;&#x80DE;&#x3078;&#x306E;&#x79FB;&#x884C;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x3092;&#x898B;&#x3064;&#x3051;&#x305F;&#x3002;&#x4E88;&#x671F;&#x305B;&#x305A;&#x3001;&#x62E1;&#x5927;, &#x9802;&#x90E8;&#x30FB;&#x57FA;&#x90E8;&#x306E;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306F;&#x5B9F;&#x8CEA;&#x7684;&#x306B;&#x9577;&#x3044; S &#x76F8;, &#x9802;&#x90E8;&#x30FB;&#x57FA;&#x90E8;&#x306E;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x751F;&#x7523;&#x3078;&#x306E;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x3088;&#x308A;&#x5C55;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x3055;&#x308C;&#x305F;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x5BFE;&#x62E1;&#x5927;&#x6BD4;&#x8F03;&#x30B2;&#x30CE;&#x30E0;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x5206;&#x6790;&#x3067;&#x306F;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x898F;&#x5236;&#x3001;DNA &#x8907;&#x88FD;&#x3001;&#x4FEE;&#x5FA9;&#x3001;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3; &#x30EA;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x91CD;&#x8981;&#x306A;&#x8981;&#x56E0;&#x306E;&#x5909;&#x5316;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30CB;&#x30E5;&#x30FC;&#x30E9;&#x30EB;&#x62E1;&#x5927;&#x5E79;&#x30FB;&#x524D;&#x99C6;&#x7D30;&#x80DE; S &#x30D5;&#x30A7;&#x30FC;&#x30BA;&#x4E2D;&#x306B;&#x305D;&#x308C;&#x3089;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x751F;&#x7523;&#x3078;&#x306E;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x3088;&#x308A;&#x591A;&#x304F;&#x306E;&#x6642;&#x9593;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30C8;&#x3055;&#x308C;&#x305F; DNA &#x306E;&#x54C1;&#x8CEA;&#x7BA1;&#x7406;&#x306B;&#x6295;&#x8CC7;&#x3059;&#x308B;&#x793A;&#x5506;&#x3057;&#x307E;&#x3059;&#x3002;

&#x795E;&#x7D4C;&#x5E79;&#x30FB;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306F;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x751F;&#x7523;&#x3078;&#x306E;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x30E1;&#x30F3;&#x30C8; S &#x76F8;&#x3092;&#x77ED;&#x304F;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002;

&#xD3EC;&#xC720;&#xB958; &#xB300;&#xB1CC; &#xD53C; &#xC9C8; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xC138;&#xD3EC; &#xC8FC;&#xAE30;&#xC758; g 1 &#xB2E8;&#xACC4;&#xB294; &#xAE38;&#xAC8C;, &#xD558;&#xC9C0;&#xB9CC; &#xADF8;&#xAC83; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xBD84;&#xBA85; &#xC788;&#xB294; &#xC2E0;&#xACBD; &#xC904;&#xAE30; &#xC54C;&#xB824;&#xC9C0;&#xACE0; &#xC870;&#xC0C1; &#xC138;&#xD3EC;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC2E0;&#xBB38;&#xC5D0; &#xC6B0;&#xB9AC; &#xC138;&#xD3EC; &#xC8FC;&#xAE30;&#xC758; &#xBD84;&#xC790; &#xB9C8;&#xCEE4; &#xBCF4;&#xB2E4;&#xB294; &#xC704;&#xCE58;&#xC5D0; &#xC758;&#xD574; &#xC2DD;&#xBCC4; &#xB41C; &#xC2E0;&#xACBD; &#xC904;&#xAE30;&#xC640; &#xC870;&#xC0C1; &#xC138;&#xD3EC;&#xC758; &#xD2B9;&#xC815; &#xD074;&#xB798;&#xC2A4;&#xC5D0; &#xB9E4;&#xAC1C; &#xBCC0;&#xC218; &#xD655;&#xC778; &#xC0C8;&#xB85C;&#xC6B4; &#xC811;&#xADFC; &#xBC29;&#xBC95;&#xC744; &#xAC1C;&#xBC1C;. &#xC6B0;&#xB9AC;&#xAC00; &#xBC1C;&#xACAC; G1 &#xAE38;&#xC5B4; &#xAD00;&#xB828; &#xB41C; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xCC98;&#xB7FC; &#xD600;&#xB05D; progenitors&#xC5D0;&#xC11C; &#xC6B4;&#xBA85; &#xC81C;&#xD55C; &#xAE30;&#xC800; (&#xC911;&#xAE09;) progenitors&#xB85C; &#xC804;&#xD658; &#xD588;&#xB2E4;. &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xAC8C;, &#xD655;&#xC7A5; &#xAF2D;&#xB300;&#xAE30;&#xC640; &#xAE30;&#xC800; progenitors &#xAF2D;&#xB300;&#xAE30;&#xC640; &#xAE30;&#xC800; progenitors &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xC0DD;&#xC0B0;&#xC744; &#xC704;&#xD574; &#xCD5C;&#xC120;&#xC744; &#xB2E4;&#xD558;&#xACE0; &#xBCF4;&#xB2E4; &#xC2E4;&#xC9C8;&#xC801;&#xC73C;&#xB85C; &#xB354; &#xC774;&#xC0C1; S &#xC0C1; &#xC804;&#xC2DC;. &#xCD5C;&#xC120;&#xC744; &#xB2E4;&#xD558;&#xACE0; &#xC870;&#xC0C1; &#xC138;&#xD3EC; &#xB300; &#xD655;&#xC7A5; &#xC2DD; &#xBD84;&#xC11D; &#xBE44;&#xAD50; &#xAC8C;&#xB188; &#xC804;&#xCCB4; &#xC720;&#xC804;&#xC790; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xC870;&#xC808;, DNA &#xBCF5;&#xC81C; &#xBC0F; &#xBCF5;&#xAD6C; &#xBC0F; &#xAC1C;&#xC7A5; &#xD558;&#xB294; chromatin&#xC758; &#xC8FC;&#xC694; &#xC694;&#xC778;&#xC758; &#xBCC0;&#xD654;&#xB97C; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xD655;&#xC7A5; &#xC2E0;&#xACBD; &#xC904;&#xAE30;&#xC640; &#xC870;&#xC0C1; &#xC138;&#xD3EC; &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xC0DD;&#xC0B0; &#xD558;&#xAE30; &#xC704;&#xD574; &#xB178;&#xB825; &#xD558;&#xB294; &#xAC83; &#xBCF4;&#xB2E4; &#xBCF5;&#xC81C; &#xB41C; DNA&#xC758; &#xD488;&#xC9C8; &#xAD00;&#xB9AC;&#xC5D0; S &#xB2E8;&#xACC4; &#xB354; &#xB9CE;&#xC740; &#xC2DC;&#xAC04;&#xC744; &#xD22C;&#xC790; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC2E0;&#xACBD; &#xC904;&#xAE30;&#xC640; &#xC870;&#xC0C1; &#xC138;&#xD3EC; &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xC0DD;&#xC0B0;&#xC5D0; &#xB300; &#xD55C; &#xC57D;&#xC18D;&#xC5D0; S &#xB2E8;&#xACC4;&#xB97C; &#xB2E8;&#xCD95;.

Durante el desarrollo de los mam&iacute;feros de corteza cerebral, la fase G1 del ciclo celular es conocida para alargar, pero ha sido claro que nervios madre y c&eacute;lulas progenitoras son afectadas. En este trabajo, desarrollamos un nuevo enfoque para determinar los par&aacute;metros del ciclo celular en clases espec&iacute;ficas de c&eacute;lulas madre y progenitoras neurales, identificadas por marcadores moleculares en lugar de ubicaci&oacute;n. Encontramos que alargar el G1 se asoci&oacute; con la transici&oacute;n de progenitores apicales de c&eacute;lulas madre similares a restricci&oacute;n de destino basales progenitores (intermedios). Inesperadamente, expansi&oacute;n apicales y basales progenitores exhiben una fase S substancialmente m&aacute;s largo que los progenitores apicales y basales, comprometida con la producci&oacute;n de la neurona. An&aacute;lisis de la expresi&oacute;n de gene de genoma comparativo de expansi&oacute;n frente a c&eacute;lulas progenitoras comprometidas revelaron cambios en factores clave de la regulaci&oacute;n del ciclo celular, replicaci&oacute;n del ADN y reparaci&oacute;n y remodelaci&oacute;n de la cromatina. Nuestros resultados sugieren que expansi&oacute;n neuronal del tallo y c&eacute;lulas progenitoras inversi&oacute;n m&aacute;s tiempo durante la fase S en control de calidad de ADN replicado que aquellos comprometidos con la producci&oacute;n de la neurona.

Las c&eacute;lulas madre y progenitoras neurales acortan la fase S en compromiso con la producci&oacute;n de la neurona.

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

A new approach to manipulate the fate of single neural stem cells in tissue.

Mammalian neocortex size primarily reflects the number and mode of divisions of neural stem and progenitor cells. Cortical stem cells (apical progenitors) switching from symmetric divisions, which expand their population, to asymmetric divisions, which generate downstream neuronal progenitors (basal progenitors), start expressing Tis21, a so-called antiproliferative/prodifferentiative gene. Tis21 encodes a small (17.5 kDa), functionally poorly characterized protein and a relatively large (2 kb), highly conserved 3' UTR. Here, we show that mice lacking the Tis21 3' UTR develop a microcephalic neocortex with fewer neurons, notably in the upper layers. This reflects a progressive decrease in basal progenitors, which in turn is due to a fraction of apical progenitors prematurely switching from asymmetric self-renewing to symmetric self-consuming divisions. This switch is caused by the markedly increased Tis21 protein level resulting from lack of microRNA-, notably miR-92-, dependent restriction of Tis21 expression. Our data show that a premature onset of consumptive neural stem cell divisions can lead to microcephaly.

3' UTR-dependent, miR-92-mediated restriction of Tis21 expression maintains asymmetric neural stem cell division to ensure proper neocortex size.

This microinjection protocol allows the manipulation and tracking of neural stem and progenitor cells in tissue at single-cell resolution. We demonstrate how to apply microinjection to organotypic brain slices obtained from mice and ferrets; however, our technique is not limited to mouse and ferret embryos, but provides a means of introducing a wide variety of membrane-impermeable molecules (e.g., nucleic acids, proteins, hydrophilic compounds) into neural stem and progenitor cells of any developing mammalian brain. Microinjection experiments are conducted by using a phase-contrast microscope equipped with epifluorescence, a transjector and a micromanipulator. The procedure normally takes ∼2 h for an experienced researcher, and the entire protocol, including tissue processing, can be performed within 1 week. Thus, microinjection is a unique and versatile method for changing and tracking the fate of a cell in organotypic slice culture. 

Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue.

Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex. 

Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion.

The evolutionary expansion of the neocortex in mammals has been linked to enlargement of the subventricular zone (SVZ) and increased proliferative capacity of basal progenitors (BPs), notably basal radial glia (bRG). The transcription factor Pax6 is known to be highly expressed in primate, but not mouse, BPs. Here, we demonstrate that sustaining Pax6 expression selectively in BP-genic apical radial glia (aRG) and their BP progeny of embryonic mouse neocortex suffices to induce primate-like progenitor behaviour. Specifically, we conditionally expressed Pax6 by in utero electroporation using a novel, Tis21-CreERT2 mouse line. This expression altered aRG cleavage plane orientation to promote bRG generation, increased cell-cycle re-entry of BPs, and ultimately increased upper-layer neuron production. Upper-layer neuron production was also increased in double-transgenic mouse embryos with sustained Pax6 expression in the neurogenic lineage. Strikingly, increased BPs existed not only in the SVZ but also in the intermediate zone of the neocortex of these double-transgenic mouse embryos. In mutant mouse embryos lacking functional Pax6, the proportion of bRG among BPs was reduced. Our data identify specific Pax6 effects in BPs and imply that sustaining this Pax6 function in BPs could be a key aspect of SVZ enlargement and, consequently, the evolutionary expansion of the neocortex. 

Sustained Pax6 Expression Generates Primate-like Basal Radial Glia in Developing Mouse Neocortex.

Midbody release from proliferative neural progenitor cells is tightly associated with the neuronal commitment of neural progenitor cells during the progression of neurogenesis in the mammalian cerebral cortex. While the central portion of the midbody, a cytoplasmic bridge between nascent daughter cells, is engulfed by one of the daughter cell by most cells in vitro, it is shown to be released into the extracellular cerebrospinal fluid (CF) in vivo in mouse embryos. Several proteins have been involved in midbody release; however, few studies have addressed the participation of the plasma membrane's lipids in this process. Here, we show by Shotgun Lipidomic analysis that phosphatydylserine (PS), among other lipids, is enriched in the released midbodies compared to lipoparticles and cellular membranes, both collected from the CF of the developing mouse embryos. Moreover, the developing mouse embryo neural progenitor cells released two distinct types of midbodies carrying either internalized PS or externalized PS on their membrane. This strongly suggests that phagocytosis and an alternative fate of released midbodies exists. HeLa cells, which are known to mainly engulf the midbody show almost no PS exposure, if any, on the outer leaflet of the midbody membrane. These results point toward that PS exposure might be involved in the selection of recipients of released midbodies, either to be engulfed by daughter cells or phagocytosed by non-daughter cells or another cell type in the developing cerebral cortex. 

Lipidome of midbody released from neural stem and progenitor cells during mammalian cortical neurogenesis.

The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here, we present genomewide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator  We use catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the  locus , which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation data and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development.

Epigenome profiling and editing of neocortical progenitor cells during development.

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.

Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low-throughput process. We developed the "Autoinjector", a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap-junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction-coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high-performance single-cell manipulation technique and will provide a powerful new platform for performing single-cell analyses in tissue for bioengineering and biophysics applications.

Robotic platform for microinjection into single cells in brain tissue.

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene  in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that  causes expansion of the primate neocortex.  expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.

Article	Year
Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 16014714	2005
Insulinoma-associated 1 has a panneurogenic role and promotes the generation and expansion of basal progenitors in the developing mouse neocortex. Neuron\| PubMed ID: 18940587	2008
miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex. Development (Cambridge, England)\| PubMed ID: 18997113	2008
Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nature communications\| PubMed ID: 21224845	2011
A new approach to manipulate the fate of single neural stem cells in tissue. Nature neuroscience\| PubMed ID: 22179113	2011
3' UTR-dependent, miR-92-mediated restriction of Tis21 expression maintains asymmetric neural stem cell division to ensure proper neocortex size. Cell reports\| PubMed ID: 24726360	2014
Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue. Nature protocols\| PubMed ID: 24762784	2014
Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion. Science (New York, N.Y.)\| PubMed ID: 25721503	2015
Sustained Pax6 Expression Generates Primate-like Basal Radial Glia in Developing Mouse Neocortex. PLoS biology\| PubMed ID: 26252244	2015
Lipidome of midbody released from neural stem and progenitor cells during mammalian cortical neurogenesis. Frontiers in cellular neuroscience\| PubMed ID: 26379497	2015
Epigenome profiling and editing of neocortical progenitor cells during development. The EMBO journal\| PubMed ID: 28765163	2017
Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7. Neuron\| PubMed ID: 29503187	2018
Robotic platform for microinjection into single cells in brain tissue. EMBO reports\| PubMed ID: 31469223	2019
Human-specific increases size and folding of primate neocortex in the fetal marmoset. Science (New York, N.Y.)\| PubMed ID: 32554627	2020

Christiane Haffner

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