modelling-zika-virus-infection-developing-human-brain-vitro-using

Modelling Zika Virus Infection of the Developing Human Brain <em>In Vitro</em> Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Novartis Institutes for BioMedical Research

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.

Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy.

The latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the proliferation of infected B lymphocytes by signaling through its binding to cellular signaling molecules. It apparently mimics members of the tumor necrosis factor receptor family, in particular, CD40, by binding a similar set of cellular molecules as does CD40. LMP-1 differs dramatically in its structure from CD40. LMP-1 has six membrane-spanning domains as opposed to CD40's one. LMP-1 also differs from CD40 in its apparent independence of a ligand for its signaling. We have examined the role of LMP-1's membrane-spanning domains in its signaling. Their substitution with six membrane-spanning domains from the LMP-2A protein of EBV yields a derivative which neither coimmunoprecipitates with LMP-1 nor signals to increase the activity of NF-kappaB as does wild-type LMP-1. These observations indicate that LMP-1 has specific sequences in its membrane-spanning domains required for these activities. LMP-1's first and sixth membrane-spanning domains have multiple leucine residues potentially similar to leucine-heptad motifs that can mediate protein-protein interactions in membranes (Gurezka et al., J. Biol. Chem. 274:9265-9270, 1999). Substitution of seven leucines in LMP-1's sixth membrane-spanning domain has no effect on its function, whereas similar substitutions in its first membrane-spanning domain yielded a derivative which aggregates as does wild-type LMP-1 but has only 3% of wild-type's ability to signal through NF-kappaB. Importantly, this derivative complements a mutant of LMP-1 with wild-type membrane-spanning domains but no carboxy-terminal signaling domain. These findings together indicate that the membrane-spanning domains of LMP-1 contribute multiple functions to its signaling.

LMP-1's transmembrane domains encode multiple functions required for LMP-1's efficient signaling.

In addition to the canonical Wnt/beta-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila [1] and the Wnt/calcium pathway in vertebrate embryos [2]. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation [3-7]. To further test this hypothesis, we have identified two zebrafish homologs of the Drosophila PCP gene prickle (pk) [8], both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/beta-catenin signaling. Like noncanonical Wnts [9-10], overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. Our results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways.

Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements.

nt signalling pathways regulate cell proliferation, cell fate and morphogenetic movements. Here, we demonstrate that the Frizzled (Fz) family of Wnt receptors, similarly to G-protein-coupled receptors (GPCRs), form specific homo- and hetero-oligomers. Two lines of evidence suggest that oligomerization occurs in the endoplasmic reticulum: first, a mutant allele of Fz4, encoding a truncated protein that is retained in the endoplasmic reticulum, is linked to the autosomal-dominant retinal degenerative disease, familial exudative vitreoretinopathy (FEVR). We show that this mutant form of Fz4 oligomerizes with wild-type Fz4, retains it in the endoplasmic reticulum and inhibits its signalling. Second, a derivative of Fz1 targeted to the endoplasmic reticulum traps wild-type Fz1 in the endoplasmic reticulum and blocks its signalling. These data support the hypothesis that oligomerization of mutant and wild-type Fz proteins occurs in the endoplasmic reticulum and may explain the genetic dominance of this FEVR allele.

Mutant Frizzled 4 associated with vitreoretinopathy traps wild-type Frizzled in the endoplasmic reticulum by oligomerization.

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21-25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence.

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells.

WNT signalling has been studied primarily in developing embryos, in which cells respond to WNTs in a context-dependent manner through changes in survival and proliferation, cell fate and movement. But WNTs also have important functions in adults, and aberrant signalling by WNT pathways is linked to a range of diseases, most notably cancer. What is the full range of diseases that involve WNT pathways? Can inhibition of WNT signalling form the basis of an effective therapy for some cancers? Could activation of WNT signalling provide new therapies for other clinical conditions? Finally, on the basis of recent experiments, might WNTs normally participate in self-renewal, proliferation or differentiation of stem cells? If so, altering WNT signalling might be beneficial to the use of stem cells for therapeutic means.

WNT and beta-catenin signalling: diseases and therapies.

The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.

Functional genomic analysis of the Wnt-wingless signaling pathway.

&#x7FFC;&#x5F0F; (Wg) Wnt &#x901A;&#x8DEF;&#x662F;&#x4E00;&#x5957;&#x6838;&#x5FC3;&#x8FDB;&#x5316;&#x4FDD;&#x6301;&#x4FE1;&#x53F7;&#x4F20;&#x5BFC;&#x901A;&#x8DEF;&#xFF0C;&#x89C4;&#x7BA1;&#x540E;&#x751F;&#x53D1;&#x5C55;&#x7684;&#x8BB8;&#x591A;&#x65B9;&#x9762;&#x4E4B;&#x4E00;&#x3002;&#x5F02;&#x5E38; Wnt &#x4FE1;&#x53F7;&#x5DF2;&#x94FE;&#x63A5;&#x5230;&#x4EBA;&#x7C7B;&#x75BE;&#x75C5;&#x3002;&#x5728;&#x672C;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x5168;&#x57FA;&#x56E0;&#x7EC4; RNA &#x5E72;&#x6270; (RNAi) &#x5C4F;&#x5E55;&#x5728;&#x679C;&#x8747;&#x7EC6;&#x80DE;&#x7B5B; Wnt &#x901A;&#x8DEF;&#x7684;&#x76D1;&#x7BA1;&#x673A;&#x6784;&#x3002;&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x4E86; 238 &#x6F5C;&#x5728;&#x7684;&#x76D1;&#x7BA1;&#x673A;&#x6784;&#xFF0C;&#x5176;&#x4E2D;&#x5305;&#x62EC;&#x5DF2;&#x77E5;&#x7684;&#x901A;&#x8DEF;&#x7684;&#x5143;&#x4EF6;&#x3001; &#x4EE5;&#x524D;&#x672A;&#x94FE;&#x63A5;&#x5230;&#x6B64;&#x901A;&#x8DEF;&#x7684;&#x529F;&#x80FD;&#x57FA;&#x56E0;&#x548C;&#x57FA;&#x56E0;&#x6CA1;&#x6709;&#x4EE5;&#x524D;&#x5206;&#x914D;&#x7684;&#x804C;&#x80FD;&#x3002;&#x4E92;&#x60E0;&#x6027;&#x6700;&#x4F73;&#x7206;&#x7834;&#x5206;&#x6790;&#x663E;&#x793A; 50%&#x7684;&#x6807;&#x8BC6;&#x5728;&#x5C4F;&#x5E55;&#x4E2D;&#x7684;&#x57FA;&#x56E0;&#x6709;&#x4EBA;&#x7C7B; orthologs&#xFF0C;&#x5176;&#x4E2D;&#x5927;&#x7EA6;&#x6709; 18%&#x662F;&#x4E0E;&#x4EBA;&#x7C7B;&#x75BE;&#x75C5;&#x76F8;&#x5173;&#x8054;&#x3002;&#x4ECE;&#x57FA;&#x4E8E;&#x5355;&#x5143;&#x683C;&#x7684;&#x5C4F;&#x5E55;&#x5728;&#x679C;&#x8747;&#x3001; &#x54FA;&#x4E73;&#x52A8;&#x7269;&#x7EC6;&#x80DE;&#x548C;&#x6591;&#x9A6C;&#x9C7C;&#x80DA;&#x80CE;&#x4E2D;&#x7684;&#x6240;&#x9009;&#x57FA;&#x56E0;&#x7684;&#x529F;&#x80FD;&#x6027;&#x5206;&#x6790;&#x8868;&#x660E;&#x8FD9;&#x4E9B;&#x57FA;&#x56E0;&#x8FDB;&#x5316;&#x6709;&#x4FDD;&#x5B88;&#x4E2D; Wnt &#x4FE1;&#x53F7;&#x7684;&#x51FD;&#x6570;&#x3002;&#x9AD8;&#x541E;&#x5410;&#x91CF; RNAi &#x5C4F;&#x5E55;&#x4E2D;&#x57F9;&#x517B;&#x7EC6;&#x80DE;&#xFF0C;&#x7136;&#x540E;&#x6309;&#x529F;&#x80FD;&#x5206;&#x6790;&#x6A21;&#x578B;&#x751F;&#x7269;&#x4F53;&#x4E2D;&#x88AB;&#x8BC1;&#x660E;&#x662F;&#x8BC6;&#x522B;&#x7684;&#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x53D1;&#x80B2;&#x4E0E;&#x75BE;&#x75C5;&#x4E2D;&#x53D7;&#x7275;&#x8FDE;&#x7684;&#x76D1;&#x7BA1;&#x673A;&#x6784;&#x7684;&#x8FC5;&#x901F;&#x7684;&#x624B;&#x6BB5;&#x3002;

Wnt &#x7FFC;&#x5F0F;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x901A;&#x8DEF;&#x7684;&#x529F;&#x80FD;&#x57FA;&#x56E0;&#x7EC4;&#x5206;&#x6790;&#x3002;

La voie de signalisation Wnt-apt&egrave;res (Wg) est un ensemble de voies de signalisation &eacute;volutivement conserv&eacute;s qui r&eacute;git de nombreux aspects du d&eacute;veloppement de m&eacute;tazoaires. La signalisation Wnt aberrante a &eacute;t&eacute; li&eacute;e &agrave; la maladie humaine. Dans la pr&eacute;sente &eacute;tude, nous avons utilis&eacute; un &eacute;cran d&#39;interf&eacute;rence (ARNi) transf&eacute;r&eacute;es RNA dans les cellules de drosophile pour d&eacute;pister les organismes de r&eacute;glementation de la signalisation Wnt. Nous avons identifi&eacute; 238 r&eacute;gulateurs potentiels, incluent des composants de la voie connue, les g&egrave;nes avec fonctions n&#39;a pas &eacute;t&eacute; li&eacute;es &agrave; cette voie et g&egrave;nes sans fonctions pr&eacute;c&eacute;demment assign&eacute;e. R&eacute;ciproque-Best-Blast analyses r&eacute;v&egrave;lent que 50 % des g&egrave;nes identifi&eacute;s dans l&#39;&eacute;cran ont des orthologues humains, dont environ 18 % sont associ&eacute;s &agrave; des maladies humaines. Les essais fonctionnels des g&egrave;nes s&eacute;lectionn&eacute;s depuis l&#39;&eacute;cran de base de cellules dans la drosophile, les cellules de mammif&egrave;res et des embryons de poisson-z&egrave;bre a d&eacute;montr&eacute; que ces g&egrave;nes ont conserv&eacute; &eacute;volutivement fonctions de signalisation Wnt. &Eacute;crans de RNAi de haut d&eacute;bit dans des cellules cultiv&eacute;es, suivies d&#39;analyses fonctionnelles dans les organismes mod&egrave;les, s&#39;av&eacute;rer un moyen rapid d&#39;identifier les responsables de la r&eacute;glementation de la signalisation des voies impliqu&eacute;es dans le d&eacute;veloppement et la maladie.

Analyse g&eacute;nomique fonctionnelle de la voie de signalisation Wnt-apt&egrave;res.

Funktionelle Genomanalyse des Wnt-Signalwegs fl&#xFC;gellosen.

Il pathway Wnt-Wingless (Wg) &egrave; uno di un set di base delle vie di segnalazione evolutivamente conservati che regola molti aspetti dello sviluppo metazoi. Segnalazione Wnt aberrante &egrave; stato collegato alla malattia umana. Nel presente studio, abbiamo utilizzato uno schermo di genomewide RNA interference (RNAi) nelle cellule di Drosophila a schermo per regolatori del pathway Wnt. Abbiamo individuato 238 regolatori potenziali, che includono componenti noti pathway, geni con funzioni non precedentemente collegate a questo percorso e geni senza funzioni assegnate in precedenza. Reciproco-Best-Blast analisi rivelano che il 50% dei geni identificati nella schermata hanno orthologs umano, di cui circa il 18% sono associato con la malattia umana. Analisi funzionale di geni selezionati dalla schermata del cell-based in drosofila, cellule di mammifero ed embrioni di zebrafish hanno dimostrato che questi geni sono evolutivamente conservato le funzioni di segnalazione Wnt. Schermi di alto-rendimento RNAi in cellule in coltura, seguite da analisi funzionale in organismi modello, dimostrano di essere un rapido mezzo di identificazione di regolatori di signaling pathways implicati nello sviluppo e malattia.

Analisi genomica funzionale della via di segnalazione Wnt-wingless.

Wnt &#x7FC5; (Wg) &#x306E;&#x7D4C;&#x8DEF;&#x306F;&#x3001;&#x5F8C;&#x751F;&#x52D5;&#x7269;&#x306E;&#x958B;&#x767A;&#x306E;&#x591A;&#x304F;&#x306E;&#x5074;&#x9762;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x9032;&#x5316;&#x7684;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306E;&#x30B3;&#x30A2;&#x306E;&#x30BB;&#x30C3;&#x30C8;&#x306E; 1 &#x3064;&#x3067;&#x3059;&#x3002;&#x7570;&#x5E38;&#x306A; wnt &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306F;&#x3001;&#x4EBA;&#x9593;&#x306E;&#x75C5;&#x6C17;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F;&#x30B2;&#x30CE;&#x30E0;&#x30EF;&#x30A4;&#x30C9; RNA &#x5E72;&#x6E09; &#xFF08;RNAi&#xFF09; &#x753B;&#x9762;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x30BB;&#x30EB;&#x3067;&#x3001;wnt &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x7D4C;&#x8DEF;&#x306E;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30BF;&#x306E;&#x753B;&#x9762;&#x306B;&#x4F7F;&#x7528;&#x3002;&#x65E2;&#x77E5;&#x306E;&#x7D4C;&#x8DEF;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306F;&#x3001;&#x4EE5;&#x524D;&#x3053;&#x306E;&#x7D4C;&#x8DEF;&#x306B;&#x306F;&#x3001;&#x30EA;&#x30F3;&#x30AF;&#x6A5F;&#x80FD;&#x3092;&#x6301;&#x3064;&#x907A;&#x4F1D;&#x5B50;&#x304A;&#x3088;&#x3073;&#x907A;&#x4F1D;&#x5B50;&#x3068;&#x4EE5;&#x524D;&#x306B;&#x5272;&#x308A;&#x5F53;&#x3066;&#x3089;&#x308C;&#x305F;&#x95A2;&#x6570;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3092;&#x542B;&#x3081;&#x308B; 238 &#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30BF;&#x3092;&#x8B58;&#x5225;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x9006;&#x6570;&#x30D9;&#x30B9;&#x30C8; &#x30D6;&#x30E9;&#x30B9;&#x30C8;&#x89E3;&#x6790;&#x753B;&#x9762;&#x3067;&#x8B58;&#x5225;&#x3057;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x306E; 50% &#x304C;&#x7D04; 18% &#x306E;&#x4EBA;&#x9593;&#x306E;&#x75C5;&#x6C17;&#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x4EBA;&#x9593;&#x306E;&#x30AA;&#x30EB;&#x30BD;&#x30ED;&#x30B0;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x3001;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x30BB;&#x30EB;&#x304A;&#x3088;&#x3073;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x80DA;&#x306E;&#x7D30;&#x80DE;&#x306B;&#x57FA;&#x3065;&#x304F;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30F3;&#x304B;&#x3089;&#x9078;&#x629E;&#x3055;&#x308C;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6A5F;&#x80FD;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x304C; wnt &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x6A5F;&#x80FD;&#x9032;&#x5316;&#x7684;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3068;&#x793A;&#x3057;&#x305F;&#x3002;&#x30CF;&#x30A4;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; RNAi &#x753B;&#x9762;&#x30E2;&#x30C7;&#x30EB;&#x6709;&#x6A5F;&#x4F53;&#x306E;&#x6A5F;&#x80FD;&#x89E3;&#x6790;&#x306B;&#x7D9A;&#x3044;&#x3066;&#x3001;&#x57F9;&#x990A;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306E;&#x958B;&#x767A;&#x3068;&#x75C5;&#x306B;&#x95A2;&#x4E0E;&#x306E;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30BF;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x306E;&#x8FC5;&#x901F;&#x306A;&#x624B;&#x6BB5;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;

Wnt wingless &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306E;&#x6A5F;&#x80FD;&#x30B2;&#x30CE;&#x30E0;&#x89E3;&#x6790;&#x3002;

Wnt &#xB0A0;&#xAC1C; (Wg) &#xD1B5;&#xB85C; metazoan &#xAC1C;&#xBC1C;&#xC758; &#xB9CE;&#xC740; &#xCE21;&#xBA74;&#xC744; &#xADDC;&#xC81C; &#xD558;&#xB294; &#xC9C4;&#xD654; &#xC801;&#xC73C;&#xB85C; &#xBCF4;&#xC874;&#xB41C; &#xC2E0;&#xD638; &#xD1B5;&#xB85C;&#xC758; &#xD575;&#xC2EC; &#xC9D1;&#xD569; &#xC911; &#xD558;&#xB098;&#xC785;&#xB2C8;&#xB2E4;. &#xD0C8; &#xC120; Wnt &#xC2E0;&#xD638;&#xC740; &#xC778;&#xAC04;&#xC758; &#xC9C8;&#xBCD1;&#xC5D0; &#xC5F0;&#xACB0; &#xB418;&#xC5C8;&#xB2E4;. &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C;&#xC5D0; &#xC6B0;&#xB9AC; Wnt &#xD1B5;&#xB85C;&#xC758; &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130;&#xC5D0; &#xB300; &#xD55C; &#xD654;&#xBA74;&#xC744; &#xCD08;&#xD30C;&#xB9AC; &#xC138;&#xD3EC;&#xC5D0; genomewide RNA &#xAC04;&#xC12D; (RNAi) &#xD654;&#xBA74;&#xC744; &#xC0AC;&#xC6A9;. &#xC54C;&#xB824;&#xC9C4;&#xB41C; &#xD1B5;&#xB85C; &#xAD6C;&#xC131; &#xC694;&#xC18C;, &#xC544;&#xB2C8; &#xC774;&#xC804;&#xC5D0; &#xD560;&#xB2F9; &#xB41C; &#xAE30;&#xB2A5;&#xC744; &#xAC00;&#xC9C4; &#xC720;&#xC804;&#xC790;, &#xC774;&#xC804;&#xC5D0;&#xC774; &#xD1B5;&#xB85C;&#xC5D0; &#xC5F0;&#xACB0; &#xD558;&#xB294; &#xAE30;&#xB2A5;&#xC744; &#xAC00;&#xC9C4; &#xC720;&#xC804;&#xC790; &#xB4F1; 238 &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xADDC;&#xC81C; &#xD30C;&#xC545;. &#xC0C1;&#xD638;-&#xBCA0;&#xC2A4;&#xD2B8;-&#xD3ED;&#xBC1C; &#xBD84;&#xC11D; &#xD654;&#xBA74;&#xC5D0;&#xC11C; &#xD655;&#xC778; &#xB41C; &#xC720;&#xC804;&#xC790;&#xC758; 50%&#xB294; &#xC57D; 18%&#xB294; &#xC778;&#xAC04;&#xC758; &#xC9C8;&#xBCD1;&#xC640; &#xAD00;&#xB828; &#xB41C; &#xC778;&#xAC04;&#xC758; orthologs &#xACF5;&#xAC1C;. &#xCD08;&#xD30C;&#xB9AC;, &#xD3EC;&#xC720;&#xB958; &#xC138;&#xD3EC;&#xC640; zebrafish &#xD0DC;&#xC544; &#xC138;&#xD3EC; &#xAE30;&#xBC18; &#xD654;&#xBA74;&#xC5D0;&#xC11C; &#xC120;&#xD0DD; &#xB41C; &#xC720;&#xC804;&#xC790;&#xC758; &#xAE30;&#xB2A5; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC774;&#xC774; &#xC720;&#xC804;&#xC790; Wnt &#xC2E0;&#xD638; &#xAE30;&#xB2A5; &#xBCF4;&#xC874; &#xC9C4;&#xD654; &#xC801;&#xC73C;&#xB85C; &#xC124;&#xBA85; &#xD588;&#xB2E4;. &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; RNAi &#xD654;&#xBA74; &#xB4A4;&#xC5D0; &#xBAA8;&#xB378; &#xC0DD;&#xBB3C; &#xAE30;&#xB2A5; &#xBD84;&#xC11D; &#xACBD;&#xC791;&#xB41C; &#xD55C; &#xC138;&#xD3EC;&#xC5D0; &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xAC1C;&#xBC1C; &#xBC0F; &#xC9C8;&#xBCD1;&#xC5D0; &#xC5F0;&#xB8E8;&#xC758; &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130;&#xB97C; &#xC2DD;&#xBCC4; &#xD558;&#xB294; &#xC2E0;&#xC18D;&#xD55C; &#xC218;&#xB2E8; &#xC99D;&#xBA85;.

&#xB0A0;&#xAC1C; Wnt &#xC2E0;&#xD638; &#xD1B5;&#xB85C;&#xC758; &#xAE30;&#xB2A5; &#xAC8C;&#xB188; &#xBD84;&#xC11D;.

La v&iacute;a de Wnt-sin alas (Wg) es uno de un conjunto b&aacute;sico de v&iacute;as de se&ntilde;alizaci&oacute;n evolutivamente conservados que regula muchos aspectos del desarrollo metazoarios. Se&ntilde;alizaci&oacute;n de Wnt aberrante se ha relacionado con enfermedades humanas. En el presente estudio, se utiliz&oacute; una pantalla de genomewide RNA de interferencia (ARNi) en c&eacute;lulas de Drosophila para reguladores de la v&iacute;a de Wnt. Identificamos 238 reguladores potenciales, que incluyen componentes de camino conocido, genes con funciones no previamente vinculadas a esta v&iacute;a y genes con ninguna funci&oacute;n previamente asignado. Rec&iacute;proco-mejor-Blast an&aacute;lisis revelan que el 50% de los genes identificados en la pantalla tienen ort&oacute;logos humanos, de los cuales aproximadamente el 18% se asocian a enfermedad humana. An&aacute;lisis funcionales de genes seleccionados desde la pantalla basadas en c&eacute;lulas en embriones de pez cebra, c&eacute;lulas de mam&iacute;fero y Drosophila demostraron que estos genes han conservado evolutivamente funciones en la se&ntilde;alizaci&oacute;n de Wnt. Pantallas de RNAi de alto rendimiento en c&eacute;lulas cultivadas, seguidas del an&aacute;lisis funcionales en organismos modelo, llegar a ser un medio r&aacute;pido de identificar reguladores de implicados en el desarrollo y la enfermedad de v&iacute;as de se&ntilde;alizaci&oacute;n.

An&aacute;lisis gen&oacute;mico funcional de la v&iacute;a de se&ntilde;alizaci&oacute;n Wnt-sin alas.

Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway.

Bili inhibits Wnt/beta-catenin signaling by regulating the recruitment of axin to LRP6.

High-throughput sequencing is gradually replacing microarrays as the preferred method for studying mRNA expression levels, providing nucleotide resolution and accurately measuring absolute expression levels of almost any transcript, known or novel. However, existing microarray data from clinical, pharmaceutical, and academic settings represent valuable and often underappreciated resources, and methods for assessing and improving the quality of these data are lacking.

Improving reliability and absolute quantification of human brain microarray data by filtering and scaling probes using RNA-Seq.

To provide a temporal framework for the genoarchitecture of brain development, we generated in situ hybridization data for embryonic and postnatal mouse brain at seven developmental stages for ∼2,100 genes, which were processed with an automated informatics pipeline and manually annotated. This resource comprises 434,946 images, seven reference atlases, an ontogenetic ontology, and tools to explore coexpression of genes across neurodevelopment. Gene sets coinciding with developmental phenomena were identified. A temporal shift in the principles governing the molecular organization of the brain was detected, with transient neuromeric, plate-based organization of the brain present at E11.5 and E13.5. Finally, these data provided a transcription factor code that discriminates brain structures and identifies the developmental age of a tissue, providing a foundation for eventual genetic manipulation or tracking of specific brain structures over development. The resource is available as the Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org).

A high-resolution spatiotemporal atlas of gene expression of the developing mouse brain.

Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression data set encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol that yields neurons suggestive of prefrontal cortex, we identified sets of genes and long noncoding RNAs that significantly change during corticogenesis and those enriched for disease-associations. Numerous alternatively spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly, and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org/) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development.

CORTECON: a temporal transcriptome analysis of in vitro human cerebral cortex development from human embryonic stem cells.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.

Genome engineering of isogenic human ES cells to model autism disorders.

As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field.

Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders.

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.

A viral strategy for targeting and manipulating interneurons across vertebrate species.

Zika virus (ZIKV) can cross the placental barrier, resulting in infection of the fetal brain and neurological defects including microcephaly. The cellular tropism of ZIKV and the identity of attachment factors used by the virus to gain access to key cell types involved in pathogenesis are under intense investigation. Initial studies suggested that ZIKV preferentially targets neural progenitor cells (NPCs), providing an explanation for the developmental phenotypes observed in some pregnancies. The AXL protein has been nominated as a key attachment factor for ZIKV in several cell types including NPCs. However, here we show that genetic ablation of AXL has no effect on ZIKV entry or ZIKV-mediated cell death in human induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids. These findings call into question the utility of AXL inhibitors for preventing birth defects after infection and suggest that further studies of viral attachment factors in NPCs are needed.

Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection.

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.

Article	Year
Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nature genetics\| PubMed ID: 12172548	2002
LMP-1's transmembrane domains encode multiple functions required for LMP-1's efficient signaling. Journal of virology\| PubMed ID: 12388716	2002
Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Current biology : CB\| PubMed ID: 12699626	2003
Mutant Frizzled 4 associated with vitreoretinopathy traps wild-type Frizzled in the endoplasmic reticulum by oligomerization. Nature cell biology\| PubMed ID: 14688793	2004
A plasmid-based system for expressing small interfering RNA libraries in mammalian cells. BMC cell biology\| PubMed ID: 15119963	2004
WNT and beta-catenin signalling: diseases and therapies. Nature reviews. Genetics\| PubMed ID: 15372092	2004
Functional genomic analysis of the Wnt-wingless signaling pathway. Science (New York, N.Y.)\| PubMed ID: 15817814	2005
Bili inhibits Wnt/beta-catenin signaling by regulating the recruitment of axin to LRP6. PloS one\| PubMed ID: 19572019	2009
Improving reliability and absolute quantification of human brain microarray data by filtering and scaling probes using RNA-Seq. BMC genomics\| PubMed ID: 24564186	2014
A high-resolution spatiotemporal atlas of gene expression of the developing mouse brain. Neuron\| PubMed ID: 24952961	2014
CORTECON: a temporal transcriptome analysis of in vitro human cerebral cortex development from human embryonic stem cells. Neuron\| PubMed ID: 24991954	2014
Genome engineering of isogenic human ES cells to model autism disorders. Nucleic acids research\| PubMed ID: 25765640	2015
Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders. Stem cell reports\| PubMed ID: 26610635	2015
A viral strategy for targeting and manipulating interneurons across vertebrate species. Nature neuroscience\| PubMed ID: 27798629	2016
Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection. Cell stem cell\| PubMed ID: 27912091	2016
A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development. Cell stem cell\| PubMed ID: 28094016	2017

Ajamete Kaykas

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