Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

University of California, Davis

Glypicans are essential modulators of cell signalling during embryogenesis. Little is known about their functions in brain development. We show here that mouse glypicans (gpc-1 to gpc-6) are differentially expressed in embryonic brains during key morphogenetic events. In gastrulating embryos, gpc-4 is the only glypican expressed in anterior visceral endoderm. During neural tube closure, gpc-4 transcripts are restricted to the anterior neural ridge and telencephalon. At this stage, gpc-1 expression shifts from trunk and head mesenchyme to neural tube. Gpc-3 mRNA appears across the ventral neural tube, then in the lamina terminalis and hypothalamus. Gpc-2 and gpc-6 transcripts are in all brain compartments. Gpc-5 is found in ventral brains as neurogenesis starts. Onset of neurogenesis also coincides with differential expression of glypican genes either in neural progenitors or in differentiating neurons. The novel expression sites of glypicans shown here contribute to the identification of signalling molecules involved in brain patterning.

Glypicans are differentially expressed during patterning and neurogenesis of early mouse brain.

Neural induction is the process that initiates nervous system development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm cells are fated to become neural in absence of instruction, and do so when bone morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the Xenopus embryo, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun-unlike the other BMP inhibitors used previously-can trigger conversion of Xenopus epidermis into neural tissue in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets, zic3 and foxD5a. Conversely, we found that zic1 can be activated by BMP inhibition in the absence of translation. Finally, Zic1 and Zic3 are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes.

BMP inhibition initiates neural induction via FGF signaling and Zic genes.

The vertebrate body plan is established in two major steps. First, mesendoderm induction singles out prospective endoderm, mesoderm and ectoderm progenitors. Second, these progenitors are spatially rearranged during gastrulation through numerous and complex movements to give rise to an embryo comprising three concentric germ layers, polarised along dorsoventral, anteroposterior and left-right axes. Although much is known about the molecular mechanisms of mesendoderm induction, signals controlling gastrulation movements are only starting to be revealed. In vertebrates, Nodal signalling is required to induce the mesendoderm, which has precluded an analysis of its potential role during the later process of gastrulation. Using time-dependent inhibition, we show that in Xenopus, Nodal signalling plays sequential roles in mesendoderm induction and gastrulation movements. Nodal activity is necessary for convergent extension in axial mesoderm and for head mesoderm migration. Using morpholino-mediated knockdown, we found that the Nodal ligands Xnr5 and Xnr6 are together required for mesendoderm induction, whereas Xnr1 and Xnr2 act later to control gastrulation movements. This control is operated via the direct regulation of key movement-effector genes, such as papc, has2 and pdgfralpha. Interestingly, however, Nodal does not appear to mobilise the Wnt/PCP pathway, which is known to control cell and tissue polarity. This study opens the way to the analysis of the genetic programme and cell behaviours that are controlled by Nodal signalling during vertebrate gastrulation. It also provides a good example of the sub-functionalisation that results from the expansion of gene families in evolution.

Distinct Xenopus Nodal ligands sequentially induce mesendoderm and control gastrulation movements in parallel to the Wnt/PCP pathway.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway.

&#x6700;&#x5408;&#x5B50;&#x57FA;&#x56E0;&#x6C89;&#x9ED8;&#x4ECB;&#x5BFC;&#x679C;&#x8747;&#x3001; &#x975E;&#x6D32;&#x722A;&#x87FE;&#x548C;&#x6591;&#x9A6C;&#x9C7C;&#x80DA;&#x80CE;&#x901A;&#x8FC7;&#x591A;&#x4E2A;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x5206;&#x88C2;&#x76F4;&#x5230; midblastula &#x8F6C; (MBT)&#x3002;&#x8FD9;&#x4E9B;&#x4E3B;&#x6218;&#x5766;&#x514B;&#x4E4B;&#x524D;, &#x8F6C;&#x5F55;&#x7684;&#x751F;&#x7269;&#xFF0C;&#x6BCF;&#x5E74;&#x5DF2;&#x786E;&#x5B9A;&#x51E0;&#x4E2A;&#x57FA;&#x56E0;&#x4F46;&#x662F;&#x5426;&#x65E9;&#x719F;&#x7279;&#x5B9A;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x91CD;&#x8981;&#x7684;&#x662F;&#x4EE5;&#x540E;&#x7684;&#x53D1;&#x5C55;&#x5DF2;&#x4E0D;&#x7ECF;&#x8FC7;&#x8BE6;&#x7EC6;&#x68C0;&#x67E5;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x627E;&#x51FA;&#x4E00;&#x7C7B;&#x7684;&#x86CB;&#x767D;&#x7F16;&#x7801; MBT &#x4E4B;&#x524D;&#x6FC0;&#x6D3B;&#x7684; VegT&#xFF0C;&#x662F;&#x65E2;&#x5B9A;&#x7684;&#x8F6C;&#x5F55;&#x8C03;&#x63A7;&#x7F51;&#x7EDC;&#x7684;&#x7EC4;&#x6210;&#x90E8;&#x5206;&#x6240;&#x9700; mesendoderm &#x611F;&#x5E94;&#x722A;&#x87FE;&#x4E2D;&#x5305;&#x62EC; Nodal &#x76F8;&#x5173;&#x914D;&#x4F53; xnr5&#x3001; xnr6&#xFF0C;&#x5C41;&#x80A1;&#x8F6C;&#x5F55;&#x56E0;&#x5B50; bix4 &#x548C; sox17&alpha; &#x7684;&#x5B55;&#x4EA7;&#x5987; T &#x7BB1;&#x56E0;&#x5B50;&#x7684;&#x6210;&#x7EE9;&#x5355;&#x3002;&#x79EF;&#x7D2F;&#x7684;&#x78F7;-Smad2 &#x7684;&#x6D3B;&#x52A8;&#x8282;&#x70B9;&#x4FE1;&#x53F7;&#x3001; &#x6807;&#x5FD7;&#x5728;&#x4E3B;&#x6218;&#x5766;&#x514B;&#x7684;&#x53D1;&#x75C5;&#x9700;&#x8981; preMBT &#x8F6C;&#x5F55;&#x548C; xnr5 &#x548C; xnr6 &#x7684;&#x6D3B;&#x52A8;&#x3002;&#x6B64;&#x5916;&#xFF0C;preMBT &#x6FC0;&#x6D3B;&#x8282;&#x70B9;&#x8DEF;&#x5F84;&#x7684; mesendodermal &#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x548C;&#x80DA;&#x80CE;&#x9635;&#x5217;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;&#x6700;&#x540E;&#xFF0C;xnr5 &#x548C; xnr6 &#x4E5F;&#x53EF;&#x4EE5;&#x6FC0;&#x6D3B;&#x81EA;&#x5DF1;&#x8868;&#x8FBE;&#x9636;&#x6BB5;&#x5288;&#x88C2;&#xFF0C;&#x6307;&#x793A; preMBT &#x8F6C;&#x5F55;&#x6709;&#x52A9;&#x4E8E;&#x524D;&#x9988;&#x7CFB;&#x7EDF;&#xFF0C;&#x5B83;&#x5141;&#x8BB8;&#x5F3A;&#x5065;&#x6D3B;&#x5316;&#x7684; Nodal &#x4FE1;&#x53F7;&#x5728;&#x4E3B;&#x6218;&#x5766;&#x514B;&#x3002;

&#x4E4B;&#x524D;&#x5728;&#x722A;&#x87FE; MBT &#x8F6C;&#x5F55;&#x7684;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#x3002;

Most zygotic genes remain transcriptionally silent in Drosophila, Xenopus, and zebrafish embryos through multiple mitotic divisions until the midblastula transition (MBT). Several genes have been identified in each of these organisms that are transcribed before the MBT, but whether precocious expression of specific mRNAs is important for later development has not been examined in detail. Here, we identify a class of protein coding transcripts activated before the MBT by the maternal T-box factor VegT that are components of an established transcriptional regulatory network required for mesendoderm induction in Xenopus laevis, including the Nodal related ligands xnr5, xnr6, and derriÃ¨re and the transcription factors bix4, and sox17Î±. Accumulation of phospho-Smad2, a hallmark of active Nodal signaling, at the onset of the MBT requires preMBT transcription and activity of xnr5 and xnr6. Furthermore, preMBT activation of the Nodal pathway is essential for mesendodermal gene expression and patterning of the embryo. Finally, xnr5 and xnr6 can also activate their own expression during cleavage stages, indicating that preMBT transcription contributes to a feed-forward system that allows robust activation of Nodal signaling at the MBT.

An essential role for transcription before the MBT in Xenopus laevis.

Plus les g&egrave;nes zygotiques restent transcriptionally silencieuses dans des embryons de drosophile, Xenopus et poisson-z&egrave;bre &agrave; travers de multiples divisions mitotiques jusqu&#39;&agrave; la transition car (MBT). Plusieurs g&egrave;nes ont &eacute;t&eacute; identifi&eacute;s dans chacun de ces organismes qui sont transcrits avant la MBT, mais si pr&eacute;coce expression des ARNm sp&eacute;cifique est importante pour le d&eacute;veloppement ult&eacute;rieur n&#39;a pas &eacute;t&eacute; examin&eacute;e en d&eacute;tail. Ici, nous identifions une classe de prot&eacute;ines codant des transcriptions activ&eacute;es avant la MBT par le facteur T-box maternels VegT qui sont des composants d&#39;un r&eacute;seau &eacute;tabli de r&eacute;gulation transcriptionnel requis pour l&#39;induction de la mesendoderm de Xenopus laevis, y compris le Nodal les ligands xnr5, xnr6 et derri&egrave;re et le bix4 de facteurs de transcription et sox17&alpha;. Accumulation de phospho-Smad2, une caract&eacute;ristique de signalisation Nodal active, d&egrave;s le d&eacute;but du Tim n&eacute;cessite preMBT transcription et l&#39;activit&eacute; de xnr5 et xnr6. En outre, preMBT l&#39;activation de la voie nodale est essentielle pour l&#39;expression des g&egrave;nes m&eacute;sendodermiques et structuration de l&#39;embryon. Enfin, xnr5 et xnr6 peuvent &eacute;galement activer leur propre expression au cours des stades du clivage, indiquant que la transcription de preMBT contribue &agrave; un syst&egrave;me de feed-forward qui permet l&#39;activation robuste du Nodal de signalisation &agrave; Tim.

Un r&ocirc;le essentiel pour la transcription avant la MBT de Xenopus laevis.

Die meisten zygotic Gene Schweigen transcriptionally in Drosophila, Xenopus und Zebrafish Embryos durch mehrere mitotische Abteilungen bis zu den midblastula &Uuml;bergang (MBT). In jedem dieser Organismen, die vor der MBT transkribiert werden mehrere Gene identifiziert wurden, aber ob fr&uuml;hreifen Ausdruck bestimmter mRNAs ist wichtig f&uuml;r die sp&auml;tere Entwicklung ist nicht im Detail untersucht worden. Hier erkennen wir eine Klasse von des Proteins Abschriften vor der MBT aktiviert werden, indem der m&uuml;tterlichen T-Box-Faktor verrottet, die Komponenten eines etablierten transkriptionelle regulatorischen Netzes sind erforderlich f&uuml;r die Mesendoderm Induktion in Xenopus Laevis, einschlie&szlig;lich der Nodal Liganden xnr5, xnr6, Derri&egrave;re und Transkription Faktoren bix4 und der sox17&alpha; im Zusammenhang. Anh&auml;ufung von Phospho-Smad2, ein Markenzeichen der aktiven Nodal Signalling, erfordert zu Beginn der MBT PreMBT Transkription und Aktivit&auml;t von xnr5 und xnr6. Dar&uuml;ber hinaus unbedingt PreMBT Aktivierung des Nodal Signalweges Mesendodermal Genexpression und Musterung des Embryos. Schlie&szlig;lich k&ouml;nnen xnr5 und xnr6 auch aktivieren ihren eigenen Ausdruck in Spaltung Phasen, darauf hinweist, dass PreMBT Transkription zu einem Feed-Forward-System tr&auml;gt, die robuste Aktivierung des Nodal erlaubt Signalisierung bei der MBT.

Eine wesentliche Rolle f&uuml;r die Transkription vor den MBT im Xenopus Laevis.

Pi&ugrave; zygotic geni rimangono transcriptionally silenziose in Drosophila, Xenopus e zebrafish embrioni attraverso molteplici divisioni mitotica finch&eacute; la transizione midblastula (MBT). Diversi geni sono stati identificati in ciascuno di questi organismi che sono trascritti prima la MBT, ma se &egrave; importante per il successivo sviluppo precoce espressione dei mRNAs specifico non &egrave; stato esaminato in dettaglio. Qui, noi identificare una classe di trascrizioni attivate prima il MBT per il fattore T-box materno VegT che sono componenti di una rete regolamentare transcriptional necessaria per induzione mesendoderm in Xenopus laevis, compreso il Nodal relativi ligandi xnr5, xnr6 e derri&egrave;re la trascrizione fattori bix4 e sox17&alpha; di codificazione della proteina. Accumulo di fosfo-Smad2, un segno distintivo di segnalazione nodale attivo, all&#39;esordio della MBT richiede trascrizione preMBT e attivit&agrave; di xnr5 e xnr6. Inoltre, preMBT attivazione del percorso nodale &egrave; essenziale per l&#39;espressione di gene mesendodermal e patterning dell&#39;embrione. Infine, xnr5 e xnr6 possono anche attivare la propria espressione durante le fasi di clivaggio, che indica che preMBT trascrizione contribuisce ad un sistema di feed-forward che consente l&#39;attivazione robusto di Nodal segnalazione presso la MBT.

Un ruolo essenziale per la trascrizione prima la MBT in Xenopus laevis.

&#x6700;&#x3082;&#x30B3;&#x30B3;&#x30E4;&#x30B7;&#x907A;&#x4F1D;&#x5B50; midblastula &#x8EE2;&#x79FB; &#xFF08;MBT&#xFF09; &#x307E;&#x3067;&#x767A;&#x73FE;&#x8ABF;&#x7BC0;&#x3092;&#x901A;&#x3058;&#x3066;&#x8907;&#x6570;&#x56DE;&#x306E;&#x5206;&#x88C2;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x3001;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x3001;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x80DA;&#x306B;&#x304A;&#x3051;&#x308B;&#x6C88;&#x9ED9;&#x3057;&#x307E;&#x3059;&#x3002;MBT &#x524D;&#x306B;&#x8EE2;&#x5199;&#x3055;&#x308C;&#x308B;&#x3053;&#x308C;&#x3089;&#x306E;&#x751F;&#x7269;&#x306E;&#x305D;&#x308C;&#x305E;&#x308C;&#x306B;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x304C;&#x540C;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x8A73;&#x7D30;&#x306B;&#x65E9;&#x719F;&#x306A;&#x767A;&#x73FE;&#x3059;&#x308B;&#x7279;&#x5B9A;&#x306E; Mrna &#x304C;&#x4EE5;&#x964D;&#x306E;&#x958B;&#x767A;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x306F;&#x691C;&#x67FB;&#x3055;&#x308C;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E; MBT &#x524D;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3001;&#x8A2D;&#x7ACB;&#x306E;&#x8EE2;&#x5199;&#x5236;&#x5FA1;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3067;&#x3042;&#x308B; VegT &#x5FC5;&#x8981;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x3001;mesendoderm &#x8A98;&#x5C0E;&#x306E;&#x305F;&#x3081;&#x306E;&#x7BC0;&#x70B9;&#x3092;&#x542B;&#x3080;&#x95A2;&#x9023;&#x914D;&#x4F4D;&#x5B50; xnr5&#x3001;xnr6&#x3001;&#x304A;&#x3088;&#x3073;&#x30C7;&#x30EA;&#x30A8;&#x30FC;&#x30EB;&#x3068;&#x8EE2;&#x5199;&#x56E0;&#x5B50; bix4 &#x304A;&#x3088;&#x3073; sox17&alpha; &#x306E;&#x6BCD;&#x4F53;&#x306E; T &#x30DC;&#x30C3;&#x30AF;&#x30B9;&#x56E0;&#x5B50;&#x306B;&#x3088;&#x3063;&#x3066;&#x8EE2;&#x5199;&#x3092;&#x7B26;&#x53F7;&#x5316;&#x306E;&#x30AF;&#x30E9;&#x30B9;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306A;&#x7BC0;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x8A8D;&#x523B;&#x6975;&#x5370;&#x30D5;&#x30A9;&#x30B9;&#x30D5;&#x30A9; Smad2 &#x306E;&#x84C4;&#x7A4D;&#x306F; MBT &#x306E;&#x958B;&#x59CB;&#x6642;&#x306B; preMBT &#x8EE2;&#x5199;&#x3068; xnr5 &#x3068; xnr6 &#x306E;&#x6D3B;&#x52D5;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x7BC0;&#x70B9;&#x306E;&#x7D4C;&#x8DEF;&#x306E; preMBT &#x6D3B;&#x6027;&#x5316;&#x306F; mesendodermal &#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x3068;&#x80DA;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x5F62;&#x6210;&#x306E;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;xnr5 &#x3068; xnr6 &#x3082;&#x5375;&#x5272;&#x671F;&#x3001;preMBT &#x8EE2;&#x5199;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x3067; MBT &#x7BC0;&#x70B9;&#x306E;&#x5805;&#x7262;&#x306A;&#x6D3B;&#x6027;&#x5316;&#x304C;&#x53EF;&#x80FD;&#x30D5;&#x30A3;&#x30FC;&#x30C9; &#x30D5;&#x30A9;&#x30EF;&#x30FC;&#x30C9; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306B;&#x5BC4;&#x4E0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x4E2D;&#x3001;&#x81EA;&#x3089;&#x306E;&#x8868;&#x73FE;&#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

&#x8EE2;&#x5199;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x306E; MBT &#x306E;&#x524D;&#x306B;&#x5FC5;&#x9808;&#x306E;&#x5F79;&#x5272;&#x3002;

Zygotic &#xC720;&#xC804;&#xC790;&#xC758; &#xAC00;&#xC7A5; midblastula &#xC804;&#xD658; (MBT)&#xAE4C;&#xC9C0; &#xC5EC;&#xB7EC; mitotic &#xBD80;&#xC11C;&#xB97C; &#xD1B5;&#xD574; &#xCD08;&#xD30C;&#xB9AC;&#xC640; Xenopus, zebrafish &#xD0DC;&#xC544;&#xC5D0; &#xC788;&#xB294; transcriptionally &#xBB35;. &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xC720;&#xC804;&#xC790;&#xB294; MBT &#xC804;&#xC5D0; &#xBCF5;&#xC0AC;&#xD560; &#xC774;&#xB7EC;&#xD55C; &#xC0DD;&#xBB3C;&#xC758; &#xAC01; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xD558;&#xC9C0;&#xB9CC; &#xD2B9;&#xC815; Mrnas&#xC758; &#xC870; &#xC219;&#xD55C; &#xC2DD; &#xC774;&#xD6C4;&#xC758; &#xAC1C;&#xBC1C;&#xC744; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xC778;&#xC9C0; &#xD558;&#xC9C0; &#xAC80;&#xC0AC; &#xC790;&#xC138;&#xD788;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xB2E8;&#xBC31;&#xC9C8; &#xCF54;&#xB529; &#xC131;&#xC801;&#xD45C;&#xB294; MBT &#xC804;&#xC5D0; &#xC124;&#xB9BD;&#xB41C; transcriptional &#xADDC;&#xC81C; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC758; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB294; VegT ligands xnr5, xnr6, &#xADF8;&#xB9AC;&#xACE0; derri&egrave;re &#xBC0F; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC694;&#xC778; bix4, &#xBC0F; sox17&alpha; &#xAD00;&#xB828; &#xD3EC;&#xD568; &#xD569;&#xB2C8;&#xB2E4; Nodal Xenopus laevis&#xC758; mesendoderm &#xC720;&#xB3C4;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xBAA8;&#xC131; T &#xAE30;&#xBCF8; &#xC694;&#xC18C;&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654;&#xC758; &#xD074;&#xB798;&#xC2A4;&#xB97C; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;. &#xD65C;&#xC131; &#xAFB8;&#xBC85;&#xAFB8;&#xBC85; &#xC878; &#xAE30; &#xC2E0;&#xD638;&#xC758; &#xD2B9;&#xC9D5; &#xC778; smad2&#xC758; &#xCD95;&#xC801;&#xC740; MBT&#xC758; &#xBC1C;&#xBCD1;&#xC5D0; preMBT &#xC804;&#xC0AC; &#xBC0F; xnr5 &#xBC0F; xnr6&#xC758; &#xD65C;&#xB3D9; &#xD544;&#xC694;&#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xAFB8;&#xBC85;&#xAFB8;&#xBC85; &#xC878; &#xAE30; &#xD1B5;&#xB85C;&#xC758; &#xD65C;&#xC131;&#xD654; preMBT mesendodermal &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604; &#xBC0F; &#xD0DC;&#xC544;&#xC758; &#xD328;&#xD130; &#xB2DD;&#xC744; &#xC704;&#xD574; &#xD544;&#xC218;&#xC801; &#xC774;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, xnr5 &#xBC0F; xnr6 preMBT &#xC804;&#xC0AC; Nodal &#xAC15;&#xB825;&#xD55C; &#xD65C;&#xC131;&#xD654;&#xD560; &#xC218; &#xC788;&#xB2E4; &#xC2E0;&#xD638;&#xB294; MBT&#xC5D0;&#xC11C; &#xD53C;&#xB4DC;-&#xD3EC;&#xC6CC;&#xB4DC; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0; &#xAE30;&#xC5EC; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xBD84;&#xC5F4; &#xB2E8;&#xACC4; &#xB3D9;&#xC548; &#xADF8;&#xB4E4;&#xC758; &#xC790;&#xC2E0;&#xC758; &#xC2DD;&#xC744; &#xD65C;&#xC131;&#xD654;&#xD560; &#xC218;&#xB3C4; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Xenopus laevis&#xC758; MBT &#xC804;&#xC5D0; &#xC804;&#xC0AC;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC778; &#xC5ED;&#xD560;.

M&aacute;s genes cig&oacute;ticos callarse transcripcionalmente en embriones de Drosophila, Xenopus y pez cebra a trav&eacute;s de m&uacute;ltiples divisiones mit&oacute;ticas hasta la transici&oacute;n midblastula (MBT). Se han identificado varios genes en cada uno de estos organismos que se transcriben antes el MBT, pero si es importante para el posterior desarrollo precoz expresi&oacute;n de los mRNAs espec&iacute;ficos no se ha examinado en detalle. Aqu&iacute;, identificamos una clase de prote&iacute;na codificaci&oacute;n transcripciones activadas antes el MBT por el factor de T-box materno VegT que son componentes de una red establecida de regulaci&oacute;n transcripcional necesaria para la inducci&oacute;n de mesendoderm en Xenopus laevis, incluidos el Nodal relacionados con ligandos xnr5, xnr6 y derri&egrave;re y factores de transcripci&oacute;n bix4 y sox17&alpha;. Acumulaci&oacute;n de fosfo-Smad2, un sello de se&ntilde;alizaci&oacute;n Nodal activo, en el inicio de los MBT requiere transcripci&oacute;n preMBT y actividad de xnr5 y xnr6. Adem&aacute;s, preMBT activaci&oacute;n de la v&iacute;a Nodal es esencial para la expresi&oacute;n del gen mesendodermal y el patr&oacute;n del embri&oacute;n. Finalmente, xnr5 y xnr6 tambi&eacute;n pueden activar su propia expresi&oacute;n durante etapas de escote, indicando que la transcripci&oacute;n preMBT contribuye a un sistema feed-forward que permite la activaci&oacute;n robusta de Nodal de se&ntilde;alizaci&oacute;n en el MBT.

Un papel esencial para la transcripci&oacute;n antes el MBT en Xenopus laevis.

Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

Ventx factors function as Nanog-like guardians of developmental potential in Xenopus.

Breaching of the cell membrane is one of the earliest and most common causes of cell injury, tissue damage, and disease. If the compromise in cell membrane is not repaired quickly, irreversible cell damage, cell death and defective organ functions will result. It is therefore fundamentally important to efficiently repair damage to the cell membrane. While the molecular aspects of single cell wound healing are starting to be deciphered, its bio-physical counterpart has been poorly investigated. Using Xenopus laevis oocytes as a model for single cell wound healing, we describe the temporal and spatial dynamics of the wound electric current circuitry and the temporal dynamics of cell membrane potential variation. In addition, we show the role of calcium influx in controlling electric current circuitry and cell membrane potential variations. (i) Upon wounding a single cell: an inward electric current appears at the wound center while an outward electric current is observed at its sides, illustrating the wound electric current circuitry; the cell membrane is depolarized; calcium flows into the cell. (ii) During cell membrane re-sealing: the wound center current density is maintained for a few minutes before decreasing; the cell membrane gradually re-polarizes; calcium flow into the cell drops. (iii) In conclusion, calcium influx is required for the formation and maintenance of the wound electric current circuitry, for cell membrane re-polarization and for wound healing.

Guillaume Luxardi

Department of Dermatology,
Institute for Regenerative Cures,
Department of Dermatology, Institute for Regenerative Cures

University of California, Davis

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Guillaume Luxardi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Dermatology,Institute for Regenerative Cures,Department of Dermatology, Institute for Regenerative Cures

University of California, Davis

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Department of Dermatology,
Institute for Regenerative Cures,
Department of Dermatology, Institute for Regenerative Cures