immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Duke University Medical Center

Mutations in the T-box transcription factor, TBX5, result in Holt-Oram syndrome (HOS), a human condition in which cardiac development is defective and forelimbs are stunted. Similarly, zebrafish tbx5 morphants and mutants (heartstrings; hst) lack pectoral fins and exhibit a persistently elongated heart that does not undergo chamber looping. Tbx5 is expressed in the developing atrium, ventricle and in pectoral fin fields, but its genetic targets are still being uncovered. In this study, evidence is provided that Tbx5 induces the expression of a specific member of the CaMK-II (the type II multifunctional Ca(2+)/calmodulin-dependent protein kinase) family; this CaMK-II is necessary for proper heart and fin development. Morphants of beta2 CaMK-II (camk2b2), but not the beta1 CaMK-II (camk2b1) paralog, exhibit bradycardia, elongated hearts and diminished pectoral fin development. Normal cardiac phenotypes can be restored by ectopic cytosolic CaMK-II expression in tbx5 morphants. Like tbx5, camk2b2 is expressed in the pectoral fin and looping heart, but this expression is diminished in both tbx5 morphant and hst embryos. Conversely, the introduction of excess Tbx5 into zebrafish embryos and mouse fibroblasts doubles CaMK-II expression. We conclude that beta CaMK-II expression and activity are necessary for proper cardiac and limb morphogenesis. These findings not only identify a morphogenic target for Ca(2+) during heart development, but support implied roles for CaMK-II in adult heart remodeling.

Tbx5-mediated expression of Ca(2+)/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis.

Intracellular calcium ion (Ca(2+)) elevation on the left side of the mouse embryonic node or zebrafish Kupffer's vesicle (KV) is the earliest asymmetric molecular event that is functionally linked to lateral organ placement in these species. In this study, Ca(2+)/CaM-dependent protein kinase (CaMK-II) is identified as a necessary target of this Ca(2+) elevation in zebrafish embryos. CaMK-II is transiently activated in approximately four interconnected cells along the anterior left wall of the KV between the six- and 12-somite stages, which is coincident with known left-sided Ca(2+) elevations. Within these cells, activated CaMK-II is observed at the surface and in clusters, which appear at the base of some KV cilia. Although seven genes encode catalytically active CaMK-II in early zebrafish embryos, one of these genes also encodes a truncated inactive variant (alphaKAP) that can hetero-oligomerize with and target active enzyme to membranes. alphaKAP, beta2 CaMK-II and gamma1 CaMK-II antisense morpholino oligonucleotides, as well as KV-targeted dominant negative CaMK-II, randomize organ laterality and southpaw (spaw) expression in lateral plate mesoderm (LPM). Left-sided CaMK-II activation was most dependent on an intact KV, the PKD2 Ca(2+) channel and gamma1 CaMK-II; however, alphaKAP, beta2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca(2+)-sensitive target in left-right asymmetry and supports a model in which membrane targeted CaMK-II hetero-oligomers in nodal cells transduce the left-sided PKD2-dependent Ca(2+) signals to the LPM.

The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry.

&#x80DE;&#x5185;&#x7684; Ca&sup2;&#x207A; &#x4FE1;&#x53F7;&#x5F71;&#x54CD; gastrulation&#x3001; &#x795E;&#x7ECF;&#x548C;&#x5668;&#x5B98;&#x53D1;&#x751F;&#x901A;&#x8FC7;&#x4ECD;&#x5728;&#x5B9A;&#x4E49;&#x7684;&#x8DEF;&#x5F84;&#x3002;&#x8BB8;&#x591A;&#x8FD9;&#x4E9B;&#x4EBA;&#x9AA8;&#x5F62;&#x6210;&#x8FC7;&#x7A0B;&#x7684;&#x4E00;&#x4E2A;&#x6F5C;&#x5728; Ca&sup2;&#x207A; &#x8C03;&#x89E3;&#x5458;&#x662F;&#x5B88;&#x6052;&#x7684;&#x9499;&#x8C03;&#x86CB;&#x767D;&#x4F9D;&#x8D56;&#x6027;&#x86CB;&#x767D;&#x6FC0;&#x9176; CaMK-&#x4E8C;&#x3002;&#x957F;&#x671F;&#x7684; Ca&sup2;&#x207A; &#x523A;&#x6FC0; CaMK &#x4E8C;&#x8F6C;&#x6362;&#x5DF2;&#x6FC0;&#x6D3B;&#x7684;&#x72B6;&#x6001;&#xFF0C;&#x5728;&#x6591;&#x9A6C;&#x9C7C;&#x4E2D;&#x68C0;&#x6D4B;&#x5230;&#x7684;&#x524D;&#x8111;&#x3001; &#x8033;&#x3001; &#x80BE;&#x3002;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x663E;&#x6027;&#x591A;&#x56CA;&#x80BE;&#x75C5;&#x5DF2;&#x94FE;&#x63A5;&#x5230;&#x7A81;&#x53D8;&#x7684; Ca&sup2;&#x207A; &#x8FDB;&#x884C;&#x8DE8;&#x56FD;&#x6FC0;&#x8FDB;&#x515A;&#x5BB6;&#x5EAD;&#x6210;&#x5458; PKD2&#xFF0C;&#x5176;&#x4E2D;&#x4E2D;&#x810A;&#x690E;&#x52A8;&#x7269;&#x6A21;&#x5F0F;&#x751F;&#x7269;&#x7684;&#x6291;&#x5236;&#x4F1A;&#x5BFC;&#x81F4;&#x80BE;&#x56CA;&#x80BF;&#x3002;PKD2 &#x7F3A;&#x548C; CaMK &#x4E8C;&#x7F3A;&#x6591;&#x9A6C;&#x9C7C;&#x80DA;&#x80CE;&#x4E0D;&#x6B63;&#x5E38;&#xFF0C;&#x5F62;&#x6210; pronephric &#x7BA1;&#x9053;&#x548C;&#x5C55;&#x89C8;&#x524D;&#x56CA;&#x80BF;&#x548C;&#x4E0D;&#x7A33;&#x5B9A;&#x7684;&#x6CC4;&#x6B96;&#x8154;&#x7EA4;&#x6BDB;&#x3002;PKD2 &#x5236;&#x6B62;&#x53EF;&#x706D;&#x6D3B; CaMK II pronephric &#x7EC6;&#x80DE;&#x548C;&#x7EA4;&#x6BDB;&#xFF0C;&#x800C;&#x4E00;&#x79EF;&#x6781; CaMK &#x4E8C;&#x6062;&#x590D; pkd2 morphants pronephric &#x98CE;&#x7BA1;&#x5F62;&#x6210;&#x3002;PKD2 &#x548C; CaMK &#x4E8C;&#x3001; &#x4E0D;&#x8DB3;&#x4E4B;&#x5904;&#x662F;&#x534F;&#x540C;&#xFF0C;&#x5728;&#x76F8;&#x540C;&#x7684;&#x9057;&#x4F20;&#x9014;&#x5F84;&#x652F;&#x6301;&#x5B83;&#x4EEC;&#x7684;&#x5B58;&#x5728;&#x3002;&#x6211;&#x4EEC;&#x5F97;&#x51FA;&#x7ED3;&#x8BBA;&#xFF1A; CaMK II PKD2 Ca&sup2;&#x207A;&#xFF0C;&#x65E2;&#x4FC3;&#x8FDB; pronephric &#x80BE;&#x7684;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x548C;&#x7A33;&#x5B9A;&#x4E3B;&#x6CC4;&#x6B96;&#x8154;&#x7EA4;&#x6BDB;&#x5173;&#x952E;&#x6548;&#x5E94;&#x3002;

CaMK II &#x662F; PKD2 &#x76EE;&#x6807;&#xFF0C;&#x4EE5;&#x4FC3;&#x8FDB; pronephric &#x80BE;&#x53D1;&#x5C55;&#x7A33;&#x5B9A;&#x7EA4;&#x6BDB;&#x3002;

Intracellular CaÂ²âº signals influence gastrulation, neurogenesis and organogenesis through pathways that are still being defined. One potential CaÂ²âº mediator of many of these morphogenic processes is CaMK-II, a conserved calmodulin-dependent protein kinase. Prolonged CaÂ²âº stimulation converts CaMK-II into an activated state that, in the zebrafish, is detected in the forebrain, ear and kidney. Autosomal dominant polycystic kidney disease has been linked to mutations in the CaÂ²âº-conducting TRP family member PKD2, the suppression of which in vertebrate model organisms results in kidney cysts. Both PKD2-deficient and CaMK-II-deficient zebrafish embryos fail to form pronephric ducts properly, and exhibit anterior cysts and destabilized cloacal cilia. PKD2 suppression inactivates CaMK-II in pronephric cells and cilia, whereas constitutively active CaMK-II restores pronephric duct formation in pkd2 morphants. PKD2 and CaMK-II deficiencies are synergistic, supporting their existence in the same genetic pathway. We conclude that CaMK-II is a crucial effector of PKD2 CaÂ²âº that both promotes morphogenesis of the pronephric kidney and stabilizes primary cloacal cilia.

CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.

Signaux intracellulaires de Ca&sup2;&#x207A; influencent la gastrulation, neurogen&egrave;se et organogen&egrave;se par des voies qui sont toujours d&eacute;finis. Un m&eacute;diateur potentiel de Ca&sup2;&#x207A; d&#39;un grand nombre de ces processus morphog&eacute;niques est CaMK-II, une kinase calmoduline-d&eacute;pendante conserv&eacute;e. Une stimulation prolong&eacute;e Ca&sup2;&#x207A; convertit CaMKII dans un &eacute;tat activ&eacute;, qui, dans le poisson-z&egrave;bre, est d&eacute;tect&eacute; dans le prosenc&eacute;phale, les oreilles et les reins. Maladie polykystique r&eacute;nale autosomique dominante a &eacute;t&eacute; li&eacute;e &agrave; des mutations dans le membre de la famille Ca&sup2;&#x207A; conducteur TRP PKD2, la r&eacute;pression dont en organismes mod&egrave;les vert&eacute;br&eacute;s se traduit par des kystes r&eacute;naux. Tant PKD2 d&eacute;ficientes et les embryons de poisson-z&egrave;bre CaMK-II-d&eacute;ficient ne forment pronephric conduits correctement et pi&egrave;ce ant&eacute;rieures kystes et d&eacute;stabilis&eacute;s cils cloacales. PKD2 suppression inactive CaMK-II dans les cellules pronephric et cils, tandis que constitutivement actif CaMKII-restaure la formation conduit pronephric dans pkd2 morphants. PKD2 et carences CaMKII sont synergiques, appuyant leur existence dans la m&ecirc;me voie g&eacute;n&eacute;tique. Nous concluons que la CaMKII est un effecteur crucial de Ca&sup2;&#x207A; PKD2 qui favorise la morphog&eacute;n&egrave;se du rein pronephric et stabilise les cils cloacales primaires.

CaMKII est une cible de PKD2 qui favorise le d&eacute;veloppement des reins pronephric et stabilise les cils.

Intrazellul&auml;re Ca&sup2;&#x207A; Signale beeinflussen die Gastrulation, Neurogenese und Organogenese durch Wege, die noch definiert werden. Eine potenzielle Ca&sup2;&#x207A; Mediator vieler dieser morphogenen Prozesse ist CaMK II, eine konservierte Calmodulin-abh&auml;ngige Proteinkinase. Verl&auml;ngerte Ca&sup2;&#x207A; Stimulierung konvertiert CaMK II in einen aktivierten Zustand, der in der Zebrafisch, im Stirnhirn, Ohr und Niere erkannt wird. Autosomal dominanten polyzystischen Nierenerkrankung ist mit Mutationen in der Durchf&uuml;hrung von Ca&sup2;&#x207A; TRP-Familienmitglied PKD2, verbunden war, die Unterdr&uuml;ckung von denen in vertebrate Modellorganismen Nieren Zysten f&uuml;hrt. PKD2-defizienten und CaMK-II-Mangel Zebrafish Embryos nicht pronephric Leitungen richtig zu bilden, und anterior Zysten und destabilisierten Kloaken Zilien. PKD2 Unterdr&uuml;ckung inaktiviert CaMK II in pronephric Zellen und Zilien, wohingegen konstitutiv aktive CaMK II pronephric Duct-Formation in pkd2 Morphants wiederherstellt. PKD2 und CaMK II-M&auml;ngel sind synergistische, deren Existenz in dem gleichen genetischen Weg unterst&uuml;tzen. Wir schlie&szlig;en, dass CaMK II eine entscheidende Effektor PKD2-Ca&sup2;&#x207A;, die Morphogenese der pronephric Niere f&ouml;rdert sowohl prim&auml;re Kloaken Zilien stabilisiert.

CaMK-II ist ein PKD2-Ziel, das pronephric Niere f&ouml;rdert und stabilisiert die Zilien.

Segnali intracellulari di Ca&sup2;&#x207A; influenzano gastrulazione, neurogenesi e organogenesi attraverso percorsi che sono ancora da definire. Un potenziale Ca&sup2;&#x207A; mediatore di molti di questi processi morfogenetico &egrave; CaMK-II, una chinasi di proteina calmodulin-dependent conservata. Prolungata stimolazione Ca&sup2;&#x207A; converte CaMK II in uno stato attivato che, nello zebrafish, viene rilevato nel proencefalo, orecchio e rene. Malattia policistica renale autosomica dominante &egrave; stata collegata alle mutazioni nel membro della famiglia Ca&sup2;&#x207A;-conducendo TRP PKD2, la soppressione dei quali in organismi modello vertebrati si traduce in cisti renali. Sia PKD2-carenti e gli embrioni di zebrafish CaMK II-carenti non riescono a formare dotti pronefrico correttamente e mostrano cisti anteriore e destabilizzato cilia cloacali. Soppressione PKD2 inattiva CaMK-II in cellule pronefrico e le ciglia, considerando che costitutivamente attive CaMK-II Ripristina formazione dotto pronefrico in morphants pkd2. PKD2 e carenze CaMK II sono sinergiche, sostenendo la loro esistenza nella stessa via genetica. Possiamo concludere che la CaMK II &egrave; un cruciale effector di PKD2 Ca&sup2;&#x207A; che promuove la morfogenesi del rene pronefrico e stabilizza ciliari cloacali.

CaMK II &egrave; un target PKD2 che promuove lo sviluppo renale pronefrico e stabilizza le ciglia.

&#x7D30;&#x80DE;&#x5185;&#x306E; Ca&sup2;&#x207A; &#x4FE1;&#x53F7;&#x539F;&#x8178;&#x9665;&#x5165;&#x3001;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x65B0;&#x751F;&#x3001;&#x307E;&#x3060;&#x5B9A;&#x7FA9;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x7D4C;&#x8DEF;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x306E;&#x5668;&#x5B98;&#x5F62;&#x6210;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x307E;&#x3059;&#x3002;1 &#x3064;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A; Ca&sup2;&#x207A; &#x30E1;&#x30C7;&#x30A3;&#x30A8;&#x30FC;&#x30BF;&#x306E;&#x3053;&#x308C;&#x3089;&#x306E;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x591A;&#x304F;&#x306E;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x30AB;&#x30EB;&#x30E2;&#x30C7;&#x30E5;&#x30EA;&#x30F3;&#x4F9D;&#x5B58;&#x6027;&#x30D7;&#x30ED;&#x30C6;&#x30A4;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x9928;&#x9577; II &#x3067;&#x3059;&#x3002;&#x9577;&#x671F; Ca&sup2;&#x207A; &#x523A;&#x6FC0;&#x9928;&#x9577; II &#x306F;&#x3001;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x3067;&#x306F;&#x3001;&#x524D;&#x8133;&#x3001;&#x8033;&#x3001;&#x814E;&#x81D3;&#x3067;&#x691C;&#x51FA;&#x3055;&#x308C;&#x308B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306E;&#x72B6;&#x614B;&#x306B;&#x5909;&#x63DB;&#x3057;&#x307E;&#x3059;&#x3002;&#x591A;&#x767A;&#x6027;&#x56A2;&#x80DE;&#x814E;&#x75BE;&#x60A3;&#x304C;&#x6291;&#x5236;&#x306E;&#x810A;&#x690E;&#x52D5;&#x7269;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x751F;&#x7269;&#x3067;&#x7D50;&#x679C;&#x304C;&#x814E;&#x56A2;&#x80DE;&#x306E;&#x5909;&#x7570; Ca&sup2;&#x207A; &#x5B9F;&#x65BD; TRP &#x306E;&#x5BB6;&#x65CF;&#x306E;&#x4E00;&#x54E1; PKD2 &#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;PKD2 &#x6B20;&#x640D;&#x3068;&#x9928;&#x9577; II &#x6B20;&#x640D;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x80DA;&#x306B;&#x304A;&#x3051;&#x308B;&#x30C0;&#x30AF;&#x30C8;&#x304C;&#x6B63;&#x3057;&#x304F;&#x5F62;&#x6210;&#x3057;&#x3001;&#x524D;&#x65B9;&#x56A2;&#x80DE;&#x3068;&#x4E0D;&#x5B89;&#x5B9A;&#x30AF;&#x30ED;&#x30A2;&#x30AB;&#x7E4A;&#x6BDB;&#x5C55;&#x793A;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3059;&#x3002;&#x6052;&#x5E38;&#x6D3B;&#x6027;&#x9928;&#x9577; II &#x306B;&#x304A;&#x3051;&#x308B;&#x30C0;&#x30AF;&#x30C8;&#x5F62;&#x6210; pkd2 morphants &#x3067;&#x5FA9;&#x5143;&#x306B;&#x5BFE;&#x3057; PKD2 &#x6291;&#x5236;&#x9928;&#x9577;-II &#x306B;&#x304A;&#x3051;&#x308B;&#x7D30;&#x80DE;&#x3068;&#x7E4A;&#x6BDB;&#x3001;&#x4E0D;&#x6D3B;&#x5316;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;PKD2 &#x3068;&#x9928;&#x9577; II &#x6B20;&#x9665;&#x540C;&#x3058;&#x907A;&#x4F1D;&#x7D4C;&#x8DEF;&#x3067;&#x305D;&#x306E;&#x5B58;&#x5728;&#x3092;&#x652F;&#x3048;&#x308B;&#x76F8;&#x4E57;&#x3001;&#x3067;&#x3059;&#x3002;&#x9928;&#x9577; II &#x91CD;&#x8981;&#x306A;&#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x30FC; PKD2 Ca&sup2;&#x207A; &#x4E21;&#x65B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x814E;&#x81D3;&#x306E;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x3001;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA; &#x30AF;&#x30ED;&#x30A2;&#x30AB;&#x7E4A;&#x6BDB;&#x3092;&#x5B89;&#x5B9A;&#x5316;&#x3055;&#x305B;&#x308B;&#x306E;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x305F;.

&#x9928;&#x9577; II &#x306B;&#x304A;&#x3051;&#x308B;&#x814E;&#x81D3;&#x958B;&#x767A;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x3001;&#x7E4A;&#x6BDB;&#x3092;&#x5B89;&#x5B9A;&#x5316; PKD2 &#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3067;&#x3059;&#x3002;

&#xC138;&#xD3EC;&#xB0B4; Ca&sup2;&#x207A; &#xC2E0;&#xD638; gastrulation, &#xC2E0;&#xC0DD; organogenesis &#xC815;&#xC758; &#xB418;&#xC5B4; &#xC788;&#xB294; &#xACBD;&#xB85C; &#xD1B5;&#xD574; &#xC601;&#xD5A5;. &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; Ca&sup2;&#x207A; &#xC911;&#xC7AC;&#xC790; morphogenic &#xC774;&#xB7EC;&#xD55C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xB9CE;&#xC740; CaMK-2, &#xBCF4;&#xC874;&#xB41C; calmodulin &#xC885;&#xC18D; &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C;. &#xC7A5;&#xAE30;&#xAC04;&#xB41C; Ca&sup2;&#x207A; &#xC790;&#xADF9; zebrafish, &#xAC1C;, &#xADC0; &#xBC0F; &#xC2E0;&#xC7A5;&#xC5D0; &#xAC10;&#xC9C0; &#xB418;&#xB294; &#xD65C;&#xC131;&#xD654; &#xB41C; &#xC0C1;&#xD0DC;&#xB85C; CaMK II&#xB97C; &#xBCC0;&#xD658; &#xD569;&#xB2C8;&#xB2E4;. Autosomal &#xC9C0;&#xBC30;&#xC801;&#xC778; polycystic &#xC2E0;&#xC7A5; &#xC9C8;&#xD658;&#xC740; &#xC2E0;&#xC7A5; cysts&#xC5D0; &#xCC99;&#xCD94; &#xBAA8;&#xD615; &#xC720;&#xAE30; &#xCCB4;&#xC5D0;&#xB294; &#xC5B5;&#xC81C; &#xACB0;&#xACFC; PKD2, Ca&sup2;&#x207A; &#xC2E4;&#xC2DC; TRP &#xAC00;&#xC871; &#xAD6C;&#xC131;&#xC6D0;&#xC5D0;&#xC11C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC5D0; &#xC5F0;&#xACB0; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBD88;&#xCDA9;&#xBD84; &#xD55C; PKD2 &#xBC0F; CaMK II &#xBD88;&#xCDA9;&#xBD84; &#xD55C; zebrafish &#xD0DC;&#xC544; pronephric &#xB355;&#xD2B8;&#xB97C; &#xC81C;&#xB300;&#xB85C; &#xD615;&#xC131; &#xD558; &#xACE0; &#xC55E;&#xCABD;&#xC5D0; cysts &#xBC0F; &#xBD88;&#xC548;&#xC815;&#xB41C; cloacal &#xC18D;&#xB208;&#xC379;&#xC744; &#xC2E4;&#xD328; &#xD569;&#xB2C8;&#xB2E4;. PKD2 &#xC5B5;&#xC81C; &#xD65C;&#xC131; constitutively CaMK II &#xBCF5;&#xC6D0; pkd2 morphants&#xC5D0;&#xC11C; pronephric &#xB355;&#xD2B8; &#xD615;&#xC131; &#xD558;&#xB294; &#xBC18;&#xBA74; pronephric &#xC140;&#xACFC; &#xC18D;&#xB208;&#xC379;, CaMK-II &#xC0DD;&#xAE34;&#xB2E4;. PKD2 &#xBC0F; CaMK II &#xACB0;&#xD568; &#xAC19;&#xC740; &#xC720;&#xC804;&#xC801; &#xD1B5;&#xB85C;&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xC874;&#xC7AC;&#xB97C; &#xC9C0; &#xC6D0;&#xD558;&#xB294; &#xC2DC;&#xB108;&#xC9C0;, &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; CaMK II&#xB294; &#xBAA8;&#xB450; pronephric &#xC2E0;&#xC7A5; morphogenesis &#xC62C;&#xB9AC;&#xACE0; &#xAE30;&#xBCF8; cloacal &#xC18D;&#xB208;&#xC379;&#xC744; &#xC548;&#xC815;&#xD654; &#xD558;&#xB294; PKD2 ca&sup2;&#x207A;&#xC758; &#xC911;&#xC694; &#xD55C; &#xC774;&#xD399;&#xD130; &#xACB0;&#xB860;.

CaMK II pronephric &#xC2E0;&#xC7A5; &#xAC1C;&#xBC1C;&#xC744; &#xCD09;&#xC9C4; &#xD558; &#xACE0; &#xC18D;&#xB208;&#xC379;&#xC744; &#xC548;&#xC815;&#xD654; &#xD558;&#xB294; PKD2 &#xD0C0;&#xAC9F;&#xC785;&#xB2C8;&#xB2E4;.

Se&ntilde;ales intracelulares de Ca&sup2;&#x207A; influyen en la neurog&eacute;nesis, gastrulaci&oacute;n y organog&eacute;nesis a trav&eacute;s de v&iacute;as que a&uacute;n est&aacute;n siendo definidos. Un mediador potencial de Ca&sup2;&#x207A; de muchos de estos procesos morfog&eacute;nicos es posible-II, una quinasa dependiente de calmodulina conservado. Prolongada estimulaci&oacute;n Ca&sup2;&#x207A; convierte CaMK-II en un estado activado que, en el pez cebra, se ha detectado en el prosenc&eacute;falo, el o&iacute;do y el ri&ntilde;&oacute;n. Enfermedad poliqu&iacute;stica renal autos&oacute;mica dominante se ha ligado a las mutaciones en el miembro de la familia del conductor Ca&sup2;&#x207A; TRP PKD2, la supresi&oacute;n de los cuales en organismos modelo vertebrados da lugar a quistes renales. PKD2 deficientes tanto en embriones de pez cebra CaMK II-deficientes no formar conductos pronephric correctamente y exhiben quistes anteriores y desestabilizadas cilios cloacales. Supresi&oacute;n de Pkd2 inactiva CaMK-II en c&eacute;lulas pronephric y cilios, mientras que constitutivamente activa CaMK-II restaura la formaci&oacute;n del conducto pronephric en pkd2 morphants. PKD2 y deficiencias CaMK-II son sin&eacute;rgicas, apoyando su existencia en la misma v&iacute;a gen&eacute;tica. Concluimos que CaMK-II es un efector crucial de PKD2 Ca&sup2;&#x207A; que promueve la morfog&eacute;nesis del ri&ntilde;&oacute;n pronephric y estabiliza los cilios primarios cloacales.

CaMK-II es un objetivo de PKD2 que promueve el desarrollo del ri&ntilde;&oacute;n pronephric y estabiliza cilios.

Zebrafish inner ear development is characterized by the crystallization of otoliths onto immotile kinocilia that protrude from sensory "hair" cells. The stereotypical formation of these sensory structures is dependent on the expression of key patterning genes and on Ca2+ signals. One potential target of Ca2+ signaling in the inner ear is the type II Ca2+/calmodulin-dependent protein kinase (CaMK-II), which is preferentially activated in hair cells, with intense activation at the base of kinocilia. In zebrafish, CaMK-II is encoded by seven genes; the expression of one of these genes (camk2g1) is enriched in hair cells. The suppression of camk2g1 expression by antisense morpholino oligonucleotides or inhibition of CaMK-II activation by the pharmacological antagonist, KN-93, results in aberrant otolith formation without preventing cilia formation. In fact, CaMK-II suppression results in additional ciliated hair cells and altered levels of Delta-Notch signaling members. DeltaA and deltaD transcripts are increased and DeltaD protein accumulates in hair cells of CaMK-II morphants, indicative of defective recycling and/or exocytosis. Our findings indicate that CaMK-II plays a critical role in the developing ear, influencing cell differentiation through extranuclear effects on Delta-Notch signaling. Continued expression and activation of CaMK-II in maculae and cristae in older embryos suggests continued roles in auditory sensory maturation and transduction.

CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling.

Autism spectrum disorder (ASD) is a heterogeneous disease in which efforts to define subtypes behaviorally have met with limited success. Hypothesizing that genetically based subtype identification may prove more productive, we resequenced the ASD-associated gene CHD8 in 3,730 children with developmental delay or ASD. We identified a total of 15 independent mutations; no truncating events were identified in 8,792 controls, including 2,289 unaffected siblings. In addition to a high likelihood of an ASD diagnosis among patients bearing CHD8 mutations, characteristics enriched in this group included macrocephaly, distinct faces, and gastrointestinal complaints. chd8 disruption in zebrafish recapitulates features of the human phenotype, including increased head size as a result of expansion of the forebrain/midbrain and impairment of gastrointestinal motility due to a reduction in postmitotic enteric neurons. Our findings indicate that CHD8 disruptions define a distinct ASD subtype and reveal unexpected comorbidities between brain development and enteric innervation.

Disruptive CHD8 mutations define a subtype of autism early in development.

Inactivating mutations in chromodomain helicase DNA binding protein 7 (CHD7) cause CHARGE syndrome, a severe multiorgan system disorder of which Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a minor feature. Recent reports have described predominantly missense CHD7 alleles in IGD patients, but it is unclear if these alleles are relevant to causality or overall genetic burden of Kallmann syndrome (KS) and normosmic form of IGD. To address this question, we sequenced CHD7 in 783 well-phenotyped IGD patients lacking full CHARGE features; we identified nonsynonymous rare sequence variants in 5.2% of the IGD cohort (73% missense and 27% splice variants). Functional analyses in zebrafish using a surrogate otolith assay of a representative set of these CHD7 alleles showed that rare sequence variants observed in controls showed no altered function. In contrast, 75% of the IGD-associated alleles were deleterious and resulted in both KS and normosmic IGD. In two families, pathogenic mutations in CHD7 coexisted with mutations in other known IGD genes. Taken together, our data suggest that rare deleterious CHD7 alleles contribute to the mutational burden of patients with both KS and normosmic forms of IGD in the absence of full CHARGE syndrome. These findings (i) implicate a unique role or preferential sensitivity for CHD7 in the ontogeny of GnRH neurons, (ii) reiterate the emerging genetic complexity of this family of IGD disorders, and (iii) demonstrate how the coordinated use of well-phenotyped cohorts, families, and functional studies can inform genetic architecture and provide insights into the developmental biology of cellular systems.

Functionally compromised CHD7 alleles in patients with isolated GnRH deficiency.

Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers.

Mutations in RAD21 disrupt regulation of APOB in patients with chronic intestinal pseudo-obstruction.

Isolated dystonia is a disorder characterized by involuntary twisting postures arising from sustained muscle contractions. Although autosomal-dominant mutations in TOR1A, THAP1, and GNAL have been found in some cases, the molecular mechanisms underlying isolated dystonia are largely unknown. In addition, although emphasis has been placed on dominant isolated dystonia, the disorder is also transmitted as a recessive trait, for which no mutations have been defined. Using whole-exome sequencing in a recessive isolated dystonia-affected kindred, we identified disease-segregating compound heterozygous mutations in COL6A3, a collagen VI gene associated previously with muscular dystrophy. Genetic screening of a further 367 isolated dystonia subjects revealed two additional recessive pedigrees harboring compound heterozygous mutations in COL6A3. Strikingly, all affected individuals had at least one pathogenic allele in exon 41, including an exon-skipping mutation that induced an in-frame deletion. We tested the hypothesis that disruption of this exon is pathognomonic for isolated dystonia by inducing a series of in-frame deletions in zebrafish embryos. Consistent with our human genetics data, suppression of the exon 41 ortholog caused deficits in axonal outgrowth, whereas suppression of other exons phenocopied collagen deposition mutants. All recessive mutation carriers demonstrated early-onset segmental isolated dystonia without muscular disease. Finally, we show that Col6a3 is expressed in neurons, with relevant mRNA levels detectable throughout the adult mouse brain. Taken together, our data indicate that loss-of-function mutations affecting a specific region of COL6A3 cause recessive isolated dystonia with underlying neurodevelopmental deficits and highlight the brain extracellular matrix as a contributor to dystonia pathogenesis.

Recessive mutations in the α3 (VI) collagen gene COL6A3 cause early-onset isolated dystonia.

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ∼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy.

Laterality in the vertebrate embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid across the embryonic node. Defects in these processes cause heterotaxy, the abnormal formation and arrangement of visceral organs that can range from complete inversion of symmetry to the selective misarrangement of organs. However, our understanding of the genetic causality for laterality defects in human beings remains relatively limited.

A human laterality disorder caused by a homozygous deleterious mutation in MMP21.

Across the span of the last 75+ years, technological and conceptual advances in genetics have found rapid implementation at the beginning of human life. From karyotype testing, to molecular cytogenetics, to gene panel testing, and now to whole exome and whole genome sequencing, each iterative expansion of our capability to acquire genetic data on the next generation has been implemented quickly in the clinical setting. In tandem, our continuously expanding ability to acquire large volumes of genetic data has generated its own challenges in terms of interpretation, clinical utility of the information, and concerns over privacy and discrimination; for the first time, we are faced with the possibility of having complete access to our genetic data from birth, if not shortly after conception. Here, we discuss the evolution of the field toward this new reality and we consider the potentially far-reaching consequences and, at present, an unclear path toward developing best practices for implementation.

Article	Year
Tbx5-mediated expression of Ca(2+)/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis. Developmental biology\| PubMed ID: 19345202	2009
The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry. Development (Cambridge, England)\| PubMed ID: 20630945	2010
CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia. Development (Cambridge, England)\| PubMed ID: 21752935	2011
CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling. Developmental biology\| PubMed ID: 23747599	2013
Disruptive CHD8 mutations define a subtype of autism early in development. Cell\| PubMed ID: 24998929	2014
Functionally compromised CHD7 alleles in patients with isolated GnRH deficiency. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25472840	2014
Mutations in RAD21 disrupt regulation of APOB in patients with chronic intestinal pseudo-obstruction. Gastroenterology\| PubMed ID: 25575569	2015
Recessive mutations in the α3 (VI) collagen gene COL6A3 cause early-onset isolated dystonia. American journal of human genetics\| PubMed ID: 26004199	2015
Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor. Human molecular genetics\| PubMed ID: 26188006	2015
Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy. Cell reports\| PubMed ID: 26257172	2015
A human laterality disorder caused by a homozygous deleterious mutation in MMP21. Journal of medical genetics\| PubMed ID: 26429889	2015
Newborn screening and the era of medical genomics. Seminars in perinatology\| PubMed ID: 26499764	2015

Ludmila Francescatto

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