4-dimensional-imaging-of-zebrafish-optic-cup-morphogenesis

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

University of Utah

&#x8F6C;&#x57FA;&#x56E0;&#x662F;&#x8BC4;&#x4F30;&#x57FA;&#x56E0;&#x529F;&#x80FD;&#x7684;&#x91CD;&#x8981;&#x5DE5;&#x5177;&#x3002;&#x6591;&#x9A6C;&#x9C7C;&#xFF0C;&#x8F6C;&#x57FA;&#x56E0;&#x5DF2;&#x7ECF;&#x906D;&#x53D7;&#x4E86;&#x4E09;&#x4E2A;&#x95EE;&#x9898;&#xFF1A; &#x5EFA;&#x7B51;&#x590D;&#x6742;&#x8868;&#x8FBE;&#x5F0F;&#x6784;&#x9020;&#x4F7F;&#x7528;&#x5E38;&#x89C4;&#x4E9A;&#x514B;&#x9686; &#xFF1B; &#x52B3;&#x52A8;&#x4F4E;&#x8F6C;&#x57FA;&#x56E0;&#x6548;&#x7387;&#xFF0C;&#x5BFC;&#x81F4;&#x9576;&#x5D4C;&#x5728;&#x77AC;&#x6001;&#x8F6C;&#x57FA;&#x56E0;&#x548C;&#x7F55;&#x89C1;&#x7684;&#x751F;&#x6B96;&#x7EC6;&#x80DE;&#x8FDB;&#x884C;&#x6CE8;&#x518C; &#xFF1B;&#x548C;&#x67E5;&#x660E;&#x751F;&#x6B96;&#x7EC6;&#x80DE;&#x8FDB;&#x884C;&#x96C6;&#x6210;&#xFF0C;&#x9664;&#x975E;&#x4F7F;&#x7528;&#x8367;&#x5149;&#x6807;&#x8BB0;&#x8F6C;&#x57FA;&#x56E0;&#x7684;&#x56F0;&#x96BE;&#x3002;Tol2kit &#x7CFB;&#x7EDF;&#x4F7F;&#x7528;&#x7279;&#x5B9A;&#x4E8E;&#x7AD9;&#x70B9;&#x57FA;&#x4E8E;&#x91CD;&#x7EC4;&#x514B;&#x9686; (&#x591A;&#x7AD9;&#x70B9;&#x7F51;&#x5173;&#x6280;&#x672F;) Tol2 &#x8F6C;&#x5EA7;&#x5B50;&#x9AA8;&#x5E72;&#x4E2D;&#x5141;&#x8BB8;&#x5FEB;&#x901F;&#x3001; &#x6A21;&#x5757;&#x5316;&#x5927;&#x4F1A; [&#x52A9;&#x5242;]-[&#x7F16;&#x7801;&#x5E8F;&#x5217;]-[3&#39; &#x6807;&#x8BB0;] &#x6784;&#x9020;&#x3002;&#x5B83;&#x5305;&#x62EC;&#x4E00;&#x4E2A;&#x76EE;&#x6807;&#x5411;&#x91CF;&#x4E0E; cmlc2:EGFP (&#x589E;&#x5F3A;&#x578B;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;) &#x8F6C;&#x57FA;&#x56E0;&#x6807;&#x8BB0;&#x548C;&#x5404;&#x5F0F;&#x5404;&#x6837;&#x7684;&#x5E7F;&#x6CDB;&#x6709;&#x7528;&#x6761;&#x76EE;&#x514B;&#x9686;&#xFF0C;&#x5305;&#x62EC;&#x70ED;&#x4F11;&#x514B;&#x86CB;&#x767D; 70 &#x548C; &beta;-&#x808C;&#x52A8;&#x86CB;&#x767D;&#x7684;&#x4FC3;&#x8FDB;&#x8005; &#xFF1B;&#x7EC6;&#x80DE;&#x8D28;&#x3001; &#x6838;&#x3001; &#x548C;&#x672C;&#x5730;&#x5316;&#x7684;&#x819C;&#x7684;&#x8367;&#x5149;&#x86CB;&#x767D;&#x8D28; &#xFF1B;&#x548C;&#x5185;&#x90E8;&#x6838;&#x7CD6;&#x4F53;&#x8FDB;&#x5165;&#x5E8F;&#x5217;&#x9A71;&#x52A8;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x5361;&#x5E26;&#x53CC;&#x987A;&#x53CD;&#x5B50;&#x8868;&#x8FBE;&#x5F0F;&#x3002;Tol2kit &#x6781;&#x5927;&#x5730;&#x65B9;&#x4FBF;&#x4E86;&#x6591;&#x9A6C;&#x9C7C;&#x8F6C;&#x57FA;&#x56E0;&#x3001; &#x7B80;&#x5316;&#x4E86;&#x5206;&#x4EAB;&#x7684;&#x514B;&#x9686;&#xFF0C;&#x5E76;&#x4F7F;&#x5927;&#x578B;&#x9879;&#x76EE;&#x68C0;&#x6D4B;&#x76D1;&#x7BA1;&#x6216;&#x7F16;&#x7801;&#x5E8F;&#x5217;&#x7684;&#x56FE;&#x4E66;&#x9986;&#x7684;&#x804C;&#x80FD;&#x3002;

Tol2kit: Tol2 &#x8F6C;&#x5EA7;&#x5B50;&#x8F6C;&#x57FA;&#x56E0;&#x6784;&#x9020;&#x7684;&#x591A;&#x7AD9;&#x70B9;&#x57FA;&#x4E8E;&#x7F51;&#x5173;&#x7684;&#x5EFA;&#x8BBE;&#x5DE5;&#x5177;&#x5305;&#x3002;

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Transg&eacute;n&egrave;se est un outil important pour &eacute;valuer la fonction des g&egrave;nes. Chez le poisson z&egrave;bre, la transg&eacute;n&egrave;se a souffert de trois probl&egrave;mes : le travail de construction constructions expression complexe utilisant subcloning conventionnel ; transg&eacute;n&egrave;se faible efficacit&eacute;, conduisant &agrave; un mosa&iuml;cisme dans transg&eacute;n&egrave;se transitoire et l&#39;incorporation de peu fr&eacute;quentes dans les cellules germinales ; et la difficult&eacute; &agrave; identifier les int&eacute;grations dans les cellules germinales &agrave; moins d&#39;utiliser un transg&egrave;ne marqueur fluorescent. Le syst&egrave;me Tol2kit utilise une in site recombinaison clonage (multisite portail technologie) pour permettre le montage rapide et modulaire de constructions [3&#39; tag] [promoteur]-[s&eacute;quence codante] - dans un squelette de transposon Tol2. Il comprend un vecteur de destination avec un marqueur de transg&eacute;n&egrave;se cmlc2:EGFP (prot&eacute;ine fluorescente verte am&eacute;lior&eacute;e) et une vari&eacute;t&eacute; de clones d&#39;entr&eacute;e largement utile, y compris les stimulateurs hsp70 et beta-actine ; prot&eacute;ines fluorescentes cytoplasmiques, nucl&eacute;aires et membrane localis&eacute;e ; ribosome interne entr&eacute;e ax&eacute;e sur la s&eacute;quence EGFP cassettes d&#39;expression bicistronique et. Le Tol2kit facilite la transgen&egrave;se zebrafish, simplifie grandement le partage des clones et permet de contr&ocirc;ler les fonctions des biblioth&egrave;ques, des s&eacute;quences de codage ou de r&eacute;glementation des projets &agrave; grande &eacute;chelle.

Le Tol2kit : un kit de construction de bas&eacute;s sur passerelle multisite pour constructions de transg&eacute;n&egrave;se Tol2 transposon.

Transgenesi sono uno strumento importante per valutare la funzione del gene. In zebrafish, transgenesi ha sofferto tre problemi: il lavoro di costruzione di costrutti espressione complessa utilizzando subcloning convenzionali; transgenesi bassa efficienza, portando ad un mosaicismo transgenetica transitoria e l&#39;incorporazione di germline infrequenti; e difficolt&agrave; di identificazione linea germinale integrazioni a meno che usando un transgene marcatore fluorescente. Il sistema Tol2kit utilizza site specific basati su ricombinazione clonazione (multisita Gateway tecnologia) che consente un assemblaggio veloce, modulare di costrutti [3&#39; tag] [promoter]-[sequenza codifica] - in una spina dorsale transposon Tol2. Esso include un vettore di destinazione con un marcatore di transgenesi cmlc2:EGFP (enhanced proteina fluorescente verde) e una serie di cloni di entrata ampiamente utili, tra cui promotori hsp70 e beta-actina; proteine fluorescenti citoplasmatiche, nucleare e membrana localizzato; e internal ribosome entry sequenza-driven EGFP cassette per l&#39;espressione di bicistronic. La Tol2kit notevolmente facilita zebrafish transgenesi, semplifica la condivisione dei cloni e permette di progetti su larga scala le funzioni delle librerie di regolamentazione o codificante di sequenze di test.

Tol2kit: un kit di costruzione basata su gateway multisito per transgenesi transposon Tol2 costruisce.

&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3067;&#x3059;&#x3002;3 &#x3064;&#x306E;&#x554F;&#x984C;&#x304B;&#x3089;&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x3092;&#x53D7;&#x3051;&#x305F;: &#x5F93;&#x6765; subcloning; &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x8907;&#x96D1;&#x306A;&#x5F0F;&#x69CB;&#x9020;&#x5EFA;&#x7BC9;&#x52B4;&#x50CD;&#x4F4E;&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x52B9;&#x7387;&#x3001;&#x30E2;&#x30B6;&#x30A4;&#x30AF;&#x904E;&#x6E21;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x307F;&#x63DB;&#x3048;&#x3067;&#x9593;&#x6B20;&#x7684;&#x306A;&#x751F;&#x6B96;&#x8A2D;&#x7ACB;&#x306B; &#x3002;&#x751F;&#x6B96;&#x7D71;&#x5408;&#x86CD;&#x5149;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x5834;&#x5408;&#x3092;&#x9664;&#x304D;&#x3001;&#x7279;&#x5B9A;&#x306E;&#x96E3;&#x3057;&#x3055;&#x3002;Tol2kit &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3067;&#x306F;&#x3001;&#x90E8;&#x4F4D;&#x7279;&#x7570;&#x7684;&#x7D44;&#x63DB;&#x3048;&#x306B;&#x57FA;&#x3065;&#x304F;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0; (&#x30DE;&#x30EB;&#x30C1;&#x30B5;&#x30A4;&#x30C8; &#x30B2;&#x30FC;&#x30C8;&#x30A6;&#x30A7;&#x30A4;&#x6280;&#x8853;) &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x30AF;&#x30A4;&#x30C3;&#x30AF;&#x3001;&#x30E2;&#x30B8;&#x30E5;&#x30FC;&#x30EB; &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA; [&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;]-[&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x756A;&#x53F7;]-[3&#39; &#x30BF;&#x30B0;] &#x69CB;&#x9020;&#x4F53;&#x306E; Tol2 &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; &#x30D0;&#x30C3;&#x30AF;&#x30DC;&#x30FC;&#x30F3;&#x3067;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x30C7;&#x30B9;&#x30C6;&#x30A3;&#x30CD;&#x30FC;&#x30B7;&#x30E7;&#x30F3; &#x30D9;&#x30AF;&#x30C8;&#x30EB; cmlc2:EGFP &#xFF08;&#x5F37;&#x5316;&#x7DD1;&#x8272;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#xFF09; &#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068; hsp70 &#x3068; &beta;-&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x306A;&#x3069;&#x5E83;&#x304F;&#x5F79;&#x306B;&#x7ACB;&#x3064;&#x30A8;&#x30F3;&#x30C8;&#x30EA; &#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x306E;&#x69D8;&#x3005; &#x306A;&#x304C;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x8CEA;&#x3001;&#x539F;&#x5B50;&#x529B;&#x3001;&#x304A;&#x3088;&#x3073;&#x819C;&#x5C40;&#x5728;&#x306E;&#x86CD;&#x5149;&#x86CB;&#x767D;&#x8CEA;&#x3002;&#x3084;&#x5185;&#x90E8;&#x30EA;&#x30DC;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x30A8;&#x30F3;&#x30C8;&#x30EA; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x99C6;&#x52D5; EGFP &#x30AB;&#x30BB;&#x30C3;&#x30C8; bicistronic &#x5F0F;&#x3002;Tol2kit &#x5927;&#x5E45;&#x306B;&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x3092;&#x5BB9;&#x6613;&#x306B;&#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x306E;&#x5171;&#x6709;&#x304C;&#x7C21;&#x5358;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3001;&#x5927;&#x898F;&#x6A21;&#x306A;&#x30D7;&#x30ED;&#x30B8;&#x30A7;&#x30AF;&#x30C8;&#x3092;&#x898F;&#x5236;&#x307E;&#x305F;&#x306F;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306E;&#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

Tol2kit: Tol2 &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3;&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x306E;&#x30DE;&#x30EB;&#x30C1;&#x30B5;&#x30A4;&#x30C8; &#x30B2;&#x30FC;&#x30C8;&#x30A6;&#x30A7;&#x30A4;&#x3092;&#x69CB;&#x7BC9;&#x30AD;&#x30C3;&#x30C8;&#x3092;&#x69CB;&#x7BC9;&#x3057;&#x307E;&#x3059;&#x3002;

Transgenesis &#xC720;&#xC804;&#xC790; &#xAE30;&#xB2A5;&#xC744; &#xD3C9;&#xAC00; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xB3C4;&#xAD6C;&#xC785;&#xB2C8;&#xB2E4;. Zebrafish, transgenesis 3 &#xBB38;&#xC81C;&#xC5D0;&#xC11C; &#xACE0;&#xD1B5; &#xD558;&#xACE0;&#xC788;&#xB2E4;: &#xAE30;&#xC874;&#xC758; subcloning;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xBCF5;&#xC7A1; &#xD55C; &#xC2DD;&#xC744; &#xAD6C;&#xBB38; &#xAC74;&#xBB3C;&#xC758; &#xB178;&#xB3D9; &#xB0AE;&#xC740; transgenesis &#xD6A8;&#xC728;&#xC131;&#xC744; &#xC774;&#xB04C;&#xC5B4; mosaicism &#xACFC;&#xB3C4; &#xC81C; &#xB2C9;&#xC5D0; &#xB4DC;&#xBB38; &#xC0DD;&#xC2DD; &#xAD00;; &#xADF8;&#xB9AC;&#xACE0; &#xD615;&#xAD11; &#xC131; &#xAC10; &#xC801; transgene &#xC0AC;&#xC6A9; &#xD558;&#xC9C0; &#xC54A;&#xB294; &#xC0DD;&#xC2DD; &#xD1B5;&#xD569; &#xC2DD;&#xBCC4;&#xC5D0; &#xC5B4;&#xB824;&#xC6C0;&#xC774; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. Tol2kit &#xC2DC;&#xC2A4;&#xD15C; Tol2 transposon &#xB4F1;&#xBF08;&#xC5D0; [&#xBC1C;&#xAE30;&#xC778;]-[&#xCF54;&#xB529; &#xC21C;&#xC11C;]-[3&#39; &#xD0DC;&#xADF8;] &#xAD6C;&#xBB38;&#xC758; &#xBE60;&#xB978;, &#xBAA8;&#xB4C8; &#xC870;&#xB9BD; &#xC788;&#xB3C4;&#xB85D; &#xC0AC;&#xC774;&#xD2B8;&#xBCC4; &#xC7AC;&#xACB0;&#xD569; &#xAE30;&#xBC18; &#xBCF5;&#xC81C; (multisite &#xAC8C;&#xC774;&#xD2B8;&#xC6E8;&#xC774; &#xAE30;&#xC220;)&#xB97C; &#xC0AC;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. &#xADF8;&#xAC83;&#xC740; &#xD3EC;&#xD568; &#xD558;&#xB294; cmlc2:EGFP (&#xD5A5;&#xC0C1; &#xB41C; &#xB179;&#xC0C9; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8;) transgenesis &#xB9C8;&#xCEE4; &#xBC0F; h s p 70 &#xBC0F; &#xBCA0;&#xD0C0; &#xAC78; &#xBC1C;&#xAE30;&#xC778;;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xB110;&#xB9AC; &#xC720;&#xC6A9;&#xD55C; &#xD56D;&#xBAA9; &#xD074;&#xB860;&#xC758; &#xB2E4;&#xC591; &#xD55C; &#xB300;&#xC0C1; &#xBCA1;&#xD130; &#xC138;&#xD3EC;&#xC9C8;, &#xD575;, &#xADF8;&#xB9AC;&#xACE0; &#xB9C9; &#xC9C0;&#xC5ED;&#xD654; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8;; &#xADF8;&#xB9AC;&#xACE0; &#xB0B4;&#xBD80; &#xB9AC;&#xBCF4;&#xC19C; &#xD56D;&#xBAA9; &#xC2DC;&#xD000;&#xC2A4; &#xAE30;&#xBC18; EGFP &#xCE74;&#xC138;&#xD2B8; bicistronic &#xC2DD;&#xC5D0; &#xB300; &#xD55C;. Tol2kit &#xD06C;&#xAC8C; zebrafish transgenesis&#xB97C; &#xC6A9;&#xC774; &#xD558; &#xAC8C;, &#xD074;&#xB860;, &#xACF5;&#xC720; &#xAC04;&#xC18C;&#xD654; &#xBC0F; &#xADDC;&#xC81C; &#xB610;&#xB294; &#xCF54;&#xB529; &#xC2DC;&#xD000;&#xC2A4;&#xC758; &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xC758; &#xAE30;&#xB2A5;&#xC744; &#xD14C;&#xC2A4;&#xD2B8; &#xD558;&#xB294; &#xB300;&#xADDC;&#xBAA8; &#xD504;&#xB85C;&#xC81D;&#xD2B8;&#xB97C; &#xAC00;&#xB2A5; &#xD558; &#xAC8C; &#xD569;&#xB2C8;&#xB2E4;.

Tol2kit: Tol2 transposon transgenesis &#xAD6C;&#xBB38;&#xC5D0; &#xB300; &#xD55C; multisite &#xAC8C;&#xC774;&#xD2B8;&#xC6E8;&#xC774; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xAC74;&#xC124; &#xD0A4;&#xD2B8;.

Transg&eacute;nesis es una herramienta importante para evaluar la funci&oacute;n del gene. En el pez cebra, transg&eacute;nesis ha sufrido tres problemas: el trabajo de construcci&oacute;n de construcciones de expresi&oacute;n compleja utilizando subclonaje convencionales; Transg&eacute;nesis baja eficiencia, llevando a mosaicismo en transg&eacute;nicos transitoria y la incorporaci&oacute;n de germline infrecuente; y dificultad en la identificaci&oacute;n del germline integraciones a menos que usando un marcador fluorescente transgen. El sistema Tol2kit utiliza espec&iacute;fica basada en la recombinaci&oacute;n clonaci&oacute;n (multisitio Gateway tecnolog&iacute;a) para permitir el montaje r&aacute;pido, modular de [promotor]-[secuencia de codificaci&oacute;n]-[3&#39; etiqueta] construcciones en un esqueleto de Tol2 transposon. Incluye un vector de destino con un marcador de transg&eacute;nesis cmlc2:EGFP (prote&iacute;na verde fluorescente mejorada) y una variedad de clones de entrada ampliamente &uacute;til, incluyendo promotores hsp70 y beta-actina; citopl&aacute;smicas, nucleares y localizada en la membrana prote&iacute;nas fluorescentes; y casetes EGFP de basadas en secuencia interna del ribosoma entrada para bicistronic expresi&oacute;n. El Tol2kit grandemente facilita la transg&eacute;nesis de pez cebra, simplifica el intercambio de clones y permite la comprobaci&oacute;n del funcionamiento de las bibliotecas de secuencias reguladoras o codificaci&oacute;n de proyectos a gran escala.

Tol2kit: construye un kit de construcci&oacute;n basados en m&uacute;ltiples sitios para Tol2 transposon transg&eacute;nesis.

Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.

A complex choreography of cell movements shapes the vertebrate eye.

The vertebrate eye acquires its basic form during the process of optic cup morphogenesis, during which the optic vesicle emerges from the brain neuroepithelium and, through a series of cell and tissue movements, transforms itself into the multilayered optic cup, containing neural retina (comprised of retinal progenitors), retinal pigmented epithelium, and the lens, which is derived from the overlying ectoderm. While great strides have been made to understand the developmental signals controlling specification, patterning, and differentiation of the optic cup, only in recent years have the cellular and molecular bases of optic cup morphogenesis begun to be unraveled. One critical component of the morphogenetic process is the extracellular matrix: the complex, glycoprotein-rich layer that surrounds the optic vesicle and lens. Though the extracellular matrix has long been visualized by classical histological techniques and postulated to play various roles in optic cup development, its functional role was uncertain. This is now beginning to change, as live imaging techniques, quantitative image analyses, molecular genetics and in vitro models yield new insights into the process of optic cup morphogenesis and the specific influences of particular extracellular matrix components and their associated signaling pathways.

Coming into focus: the role of extracellular matrix in vertebrate optic cup morphogenesis.

The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis.

High-resolution analysis of central nervous system expression patterns in zebrafish Gal4 enhancer-trap lines.

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1(UW1)) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1(UW1) mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis.

Establishment of precise three-dimensional tissue structure is vital for organ function. In the visual system, optic fissure and stalk morphogenesis is a crucial yet poorly understood process, disruptions of which can lead to coloboma, a birth defect causing visual impairment. Here, we use four-dimensional imaging, cell tracking, and molecular genetics in zebrafish to define the cell movements underlying normal optic fissure and stalk formation. We determine how these events are disrupted in a coloboma model in which the Hedgehog (Hh) receptor  is lost, resulting in overactive Hh signaling. In the  mutant, cells exhibit defective motile behaviors and morphology. Cells that should contribute to the fissure do not arrive at their correct position, and instead contribute to an ectopically large optic stalk. Our results suggest that overactive Hh signaling, through overexpression of downstream transcriptional targets, impairs cell motility underlying optic fissure and stalk formation, via non-cell-autonomous and cell-autonomous mechanisms. More broadly, our cell motility and morphology analyses provide a new framework for studying other coloboma-causing mutations that disrupt optic fissure or stalk formation.

Hedgehog signaling regulates cell motility and optic fissure and stalk formation during vertebrate eye morphogenesis.

Epithelial wound healing requires a complex orchestration of cellular rearrangements and movements to restore tissue architecture and function after injury. While it is well known that mechanical forces can affect tissue morphogenesis and patterning, how the biophysical cues generated after injury influence cellular behaviors during tissue repair is not well understood. Using time-lapse confocal imaging of epithelial tissues in living zebrafish larvae, we provide evidence that localized increases in cellular crowding during wound closure promote the extrusion of nonapoptotic cells via mechanically regulated stretch-activated ion channels (SACs). Directed cell migration toward the injury site promoted rapid changes in cell number and generated shifts in tension at cellular interfaces over long spatial distances. Perturbation of SAC activity resulted in failed extrusion and increased proliferation in crowded areas of the tissue. Together, we conclude that localized cell number plays a key role in dictating cellular behaviors that facilitate wound closure and tissue repair.

Cellular crowding influences extrusion and proliferation to facilitate epithelial tissue repair.

Epithelial morphogenesis, a fundamental aspect of development, generates 3-dimensional tissue structures crucial for organ function. Underlying morphogenetic mechanisms are, in many cases, poorly understood, but mutations that perturb organ development can affect epithelial cell shape and orientation - difficult features to quantify in three dimensions. The basic structure of the eye is established via epithelial morphogenesis: in the embryonic optic cup, the retinal progenitor epithelium enwraps the lens. We previously found that loss of the extracellular matrix protein laminin-alpha1 (lama1) led to mislocalization of apical polarity markers and apparent misorientation of retinal progenitors. We sought to visualize and quantify this phenotype, and determine whether loss of the apical polarity determinant pard3 might rescue the phenotype. To this end, we developed LongAxis, a MATLAB-based program optimized for the retinal progenitor neuroepithelium. LongAxis facilitates 3-dimensional cell segmentation, visualization, and quantification of cell orientation and morphology. Using LongAxis, we find that retinal progenitors in the lama1 optic cup are misoriented and slightly less elongated. In the lama1;MZpard3 double mutant, cells are still misoriented, but larger. Therefore, loss of pard3 does not rescue loss of lama1, and in fact uncovers a novel cell size phenotype. LongAxis enables population-level visualization and quantification of retinal progenitor cell orientation and morphology. These results underscore the importance of visualizing and quantifying cell orientation and shape in three dimensions within the retina.

LongAxis: A MATLAB-based program for 3D quantitative analysis of epithelial cell shape and orientation.

Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.

Optic cup morphogenesis requires neural crest-mediated basement membrane assembly.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Efficient targeted integration directed by short homology in zebrafish and mammalian cells.

The basic structure of the eye, which is crucial for visual function, is established during the embryonic process of optic cup morphogenesis. Molecular pathways of specification and patterning are integrated with spatially distinct cell and tissue shape changes to generate the eye, with discrete domains and structural features: retina and retinal pigment epithelium enwrap the lens, and the optic fissure occupies the ventral surface of the eye and optic stalk. Interest in the underlying cell biology of eye morphogenesis has led to a growing body of work, combining molecular genetics and imaging to quantify cellular processes such as adhesion and actomyosin activity. These studies reveal that intrinsic machinery and spatiotemporally specific extrinsic inputs collaborate to control dynamics of cell movements and morphologies. Here we consider recent advances in our understanding of eye morphogenesis, with a focus on the mechanics of eye formation throughout vertebrate systems, including insights and potential opportunities using organoids, which may provide a tractable system to test hypotheses from embryonic models.

Kristen M. Kwan

Department of Human Genetics

University of Utah

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis