Understanding Early Organogenesis Using a Simplified <em>In Situ</em> Hybridization Protocol in <em>Xenopus</em>

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

University of Western Ontario

Hospital for Sick Children

We have isolated the Xenopus homologue of cardiac myosin-binding protein C ( cMyBP-C) and describe its expression during early embryogenesis. cMyBP-C is expressed in both somites and heart at the time these tissues differentiate. Expression in the somites declines in older tadpoles. There is a high degree of conservation in residues where mutations causing hypertrophic cardiomyopathy have been identified in humans.

Developmental expression of cardiac myosin-binding protein C in Xenopus.

We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.

Expression of muscle LIM protein during early development in Xenopus laevis.

The region with the potential to form the heart has traditionally been called the heart field. This region can be approximated by, but is not identical to, the expression domain of the early cardiac gene Nkx2.5. The region expressing Nkx2.5 does not change in size, although there are major shape changes and a subdivision of the region into non-myogenic and myogenic lineages. Using a variety of embryo manipulations, we have sought to determine whether cellular interactions could change the size of the initial Nkx2.5-expressing region and thus change the size of the heart. We have shown that if the heart is isolated from the dorsal half of the embryo, the volume of tissue expressing myocardial differentiation markers increases, indicating that signals restricting the size of the heart come from the dorsal side. Despite the change in myocardial volume, the non-myogenic heart lineages are still present. The ability of dorsal tissues to restrict the size of the heart is further demonstrated by fusing two Xenopus embryos shortly after gastrulation, generating twinned embryos where the heart of one embryo would develop adjacent to different tissues of the second embryo. The final size of the differentiated heart was markedly reduced if it developed in close proximity to the dorso-anterior surface of the head but not if it developed adjacent to the flank or belly. In all cases, the manipulations that restricted the size of the myocardium also restricted the expression of Nkx2.5 and GATA-4, both key regulatory genes in the cardiogenic pathway. These results provide evidence for a model in which signals from dorso-anterior tissues restrict the size of the heart after gastrulation but before neural fold closure.

Regulation of heart size in Xenopus laevis.

The role of thyroid hormone in Xenopus metamorphosis is particularly well understood as it plays an essential role in that process. However, recent evidence suggests that thyroid hormone may play an earlier role in amphibian embryogenesis. We demonstrate that Xenopus thyroid hormone receptor beta (XTR beta) is expressed shortly after neural fold closure, and that its expression is localized to the developing retina. Retinoid X receptor gamma (RXR gamma), a potential dimerization partner for XTR beta, was also found to be expressed in the retina at early stages, and at later stages RXR gamma was also expressed in the liver diverticulum. Addition of either thyroid hormone or 9-cis retinoic acid, the ligands for XTR beta and RXR gamma, respectively, did not alter the expression of their receptors. However, the addition of thyroid hormone and 9-cis retinoic acid did alter rhodopsin mRNA expression. Addition of thyroid hormone generates a small expansion of the rhodopsin expression domain. When 9-cis retinoic acid or a combination of thyroid hormone and 9-cis retinoic acid was administered, there was a decrease in the expression domain of rhodopsin in the developing retina. These results provide evidence for an early role for XTR beta and RXR gamma in the developing Xenopus retina.

Early expression of thyroid hormone receptor beta and retinoid X receptor gamma in the Xenopus embryo.

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.

Retinoic acid signaling is essential for formation of the heart tube in Xenopus.

The lateral plate mesoderm (LPM) lines the body cavities, gives rise to the heart and circulatory system and is responsible for patterning the underlying endoderm. We describe gene expression domains within the lateral plate mesoderm of the neurula stage Xenopus embryo that demonstrate a marked anterior posterior pattern in that tissue. FoxF1 and Nkx-2.5 are expressed in the anterior LPM, Hand1 in the middle and Xsal-1 in the posterior LPM. Since retinoic acid is known to pattern many tissues during development, and RALDH2, the enzyme primarily responsible for retinoic acid synthesis, is expressed in the anterior and dorsal LPM, we hypothesized that retinoic acid is necessary for correct patterning of the LPM. Exposure to exogenous retinoic acid during neurulation led to an expansion of the anterior and middle expression domains and a reduction of the posterior domain whereas exposure to a retinoic acid antagonist resulted in smaller anterior and middle expression domains. Furthermore, inhibition of RALDH2, which should decrease endogenous RA levels, caused a reduction of anterior domains indicating that endogenous RA is necessary for regulating their size. After altering retinoic acid signaling in a temporally restricted window, the displaced anterior-posterior pattern is maintained until gut looping, as demonstrated by permanently altered Hand1, FoxF1, xHoxC-10, and Pitx2 expression domains. We conclude that the broad expression domains of key transcription factors demonstrate a novel anterior-posterior pattern within the LPM and that retinoic acid can regulate the size of these domains in a coordinated manner.

Retinoic acid regulates anterior-posterior patterning within the lateral plate mesoderm of Xenopus.

&#x4E86;&#x89E3;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x6CBB;&#x7597;&#x672A;&#x6765;&#x7684;&#x8BBE;&#x8BA1;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;&#x867D;&#x7136;&#x89C6;&#x9EC4;&#x9178; (RA) &#x662F;&#x6700;&#x6709;&#x529B;&#x7684;&#x5C0F;&#x5206;&#x5B50;&#x540C;&#x6E90;&#x6027;&#x9AA8;&#x9ABC;&#x808C;&#x6210;&#x808C;&#x7EC6;&#x80DE;&#x4E2D;&#xFF0C;&#x9636;&#x6BB5;&#xFF0C;&#x5176;&#x4F5C;&#x7528;&#x673A;&#x7406;&#x5C1A;&#x672A;&#x9610;&#x660E;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x4E0E;&#x5176;&#x4ED6;&#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x7684;&#x523A;&#x6FC0;&#x6216;&#x6291;&#x5236;&#x6210;&#x808C;&#x7684; RA &#x7684;&#x4EA4;&#x70B9; (&#x5982; Wnt &#x548C; BMP4&#xFF0C;&#x5206;&#x522B;) &#x662F;&#x672A;&#x77E5;&#x7684;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x7684;&#x76EE;&#x7684;&#x662F;&#x5BA1;&#x67E5;&#x7684;&#x5206;&#x5B50;&#x673A;&#x5236;&#x7684; RA &#x589E;&#x5F3A;&#x9AA8;&#x9ABC;&#x808C;&#x6210;&#x808C;&#x548C; Wnt &#x548C; BMP4 &#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#x671F;&#x95F4; P19 &#x6216;&#x9F20;&#x6807; (ES) &#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x7684;&#x4FE1;&#x53F7;&#x3002;

&#x89C6;&#x9EC4;&#x9178;&#x53EF;&#x589E;&#x5F3A;&#x9AA8;&#x9ABC;&#x808C;&#x8089;&#x7956;&#x5F62;&#x6210;&#xFF0C;&#x5E76;&#x8DF3;&#x8FC7;&#x9AA8;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x86CB;&#x767D; 4 &#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x5360;&#x4E3B;&#x5BFC;&#x5730;&#x4F4D;&#x7684;&#x6D88;&#x6781;&#x8FDE;&#x73AF;&#x3002;

Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation.

Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin.

La diff&#xE9;renciation des cellules souches compr&#xE9;hension est essentielle pour la conception future des th&#xE9;rapies cellulaires. Bien que l&#x26;#39;acide r&#xE9;tino&#xEF;que (RA) est la mol&#xE9;cule la plus puissante petite activateur de myogen&#xE8;se squelettique dans les cellules souches, le stade et le m&#xE9;canisme de sa fonction n&#x26;#39;a pas encore &#xE9;t&#xE9; &#xE9;lucid&#xE9;. En outre, l&#x26;#39;intersection de la polyarthrite rhumato&#xEF;de avec les autres voies de signalisation qui stimulent ou inhibent la myogen&#xE8;se (comme Wnt et BMP4, respectivement) est inconnu. Ainsi, dans le but de cette &#xE9;tude est d&#x26;#39;examiner les m&#xE9;canismes mol&#xE9;culaires par lesquels RA am&#xE9;liore myogen&#xE8;se squelettique et interagit avec de signalisation Wnt et BMP4 au cours P19 ou embryonnaire de la souris la diff&#xE9;renciation des cellules souches (ES).

L&#x26;#39;acide r&#xE9;tino&#xEF;que am&#xE9;liore squelettique Formation prog&#xE9;nitrices musculaires et contourne l&#x26;#39;inhibition par la prot&#xE9;ine morphog&#xE9;n&#xE9;tique osseuse 4, mais pas dominant n&#xE9;gatif de b&#xEA;ta-cat&#xE9;nine

Verst&auml;ndnis Stammzelle Differenzierung ist notwendig f&uuml;r die zuk&uuml;nftige Gestaltung von Zelltherapien. Zwar Retins&auml;ure (RA) die st&auml;rkste niedermolekularer Vergr&ouml;&szlig;erer Skelett Myogenese in Stammzellen, hat die B&uuml;hne und den Mechanismus ihrer Funktion nicht noch gekl&auml;rt. Weitere, die Schnittmenge der RA mit anderen Signalwege, die stimulieren oder hemmen Myogenese (z. B. Wnt und BMP4, beziehungsweise) ist unbekannt. Somit ist der Zweck dieser Studie untersuchen die molekularen Mechanismen, durch den RA erh&ouml;ht Skelett Myogenese und interagiert mit Wnt und Bmp4, Signalisierung w&auml;hrend der embryonalen Stammzellen (ES)-Zelldifferenzierung P19 oder Maus.

Retins&auml;ure verbessert die Skelettmuskulatur Stammvater Bildung und umgeht die Hemmung durch Knochen morphogenetische Protein 4 aber nicht dominierende negative Beta-Catenin.

Differenziazione delle cellule staminali di comprensione &egrave; essenziale per il futuro design delle terapie cellulari. Mentre l&#39;acido retinoico (RA) &egrave; il pi&ugrave; potente rinforzatore di piccola molecola del myogenesis scheletrica in cellule staminali, il palco e il meccanismo della sua funzione non &egrave; ancora stata chiarita. Ulteriormente, l&#39;intersezione di RA con altre vie di segnalazione che stimolare o inibire myogenesis (ad esempio Wnt e BMP4, rispettivamente) &egrave; sconosciuta. Cos&igrave;, lo scopo di questo studio &egrave; quello di esaminare i meccanismi molecolari che RA migliora myogenesis scheletrico e interagisce con il Wnt e BMP4 segnalazione durante il mouse o P19 differenziazione delle cellule staminali embrionali (ES).

L&#39;acido retinoico aumenta la formazione di muscolo scheletrico progenitore e bypassa l&#39;inibizione da bone morphogenetic protein 4 ma non dominante negativo beta-catenina.

&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x3053;&#x3068;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x7642;&#x6CD5;&#x306E;&#x5C06;&#x6765;&#x8A2D;&#x8A08;&#x304C;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;&#x3002;&#x30EC;&#x30C1;&#x30CE;&#x30A4;&#x30F3;&#x9178;&#xFF08;RA&#xFF09;&#x306F;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x9AA8;&#x683C;&#x7B4B;&#x5F62;&#x6210;&#x306E;&#x6700;&#x3082;&#x5F37;&#x529B;&#x306A;&#x5C0F;&#x5206;&#x5B50;&#x30A8;&#x30F3;&#x30CF;&#x30F3;&#x30B5;&#x30FC;&#x3067;&#x3059;&#x304C;&#x3001;&#x30B9;&#x30C6;&#x30FC;&#x30B8;&#x3068;&#x305D;&#x306E;&#x6A5F;&#x80FD;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x307E;&#x3060;&#x89E3;&#x660E;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#xFF08;&#x4F8B;&#x3048;&#x3070;&#x3001;Wnt&#x304A;&#x3088;&#x3073;BMP4&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x306E;&#x3088;&#x3046;&#x306A;&#xFF09;&#x7B4B;&#x5F62;&#x6210;&#x3092;&#x4FC3;&#x9032;&#x307E;&#x305F;&#x306F;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x4ED6;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x3068;RA&#x3068;&#x306E;&#x4EA4;&#x70B9;&#x306F;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308B;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x672C;&#x7814;&#x7A76;&#x306E;&#x76EE;&#x7684;&#x306F;&#x3001;RA&#x306F;&#x3001;&#x9AA8;&#x683C;&#x7B4B;&#x5F62;&#x6210;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x3001;P19&#x307E;&#x305F;&#x306F;&#x30DE;&#x30A6;&#x30B9;&#x80DA;&#x6027;&#x5E79;&#xFF08;ES&#xFF09;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x4E2D;&#x306B;Wnt&#x304A;&#x3088;&#x3073;BMP4&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x3068;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3059;&#x308B;&#x5206;&#x5B50;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x8ABF;&#x3079;&#x308B;&#x3053;&#x3068;&#x3067;&#x3059;&#x3002;

&#x30EC;&#x30C1;&#x30CE;&#x30A4;&#x30F3;&#x9178;&#x306F;&#x3001;&#x30C9;&#x30DF;&#x30CA;&#x30F3;&#x30C8;&#x30CD;&#x30AC;&#x30C6;&#x30A3;&#x30D6;&#x3B2;-&#x30AB;&#x30C6;&#x30CB;&#x30F3;&#x9AA8;&#x683C;&#x7B4B;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x5F62;&#x6210;&#x3092;&#x5F37;&#x5316;&#x3057;&#x3001;&#x9AA8;&#x5F62;&#x6210;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;4&#x306B;&#x3088;&#x3063;&#x3066;&#x963B;&#x5BB3;&#x3092;&#x30D0;&#x30A4;&#x30D1;&#x30B9;&#x3057;&#x307E;&#x305B;&#x3093;

&#xC774;&#xD574; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654; &#xC138;&#xD3EC; &#xCE58;&#xB8CC;&#xC758; &#xBBF8;&#xB798; &#xC124;&#xACC4;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;. Retinoic &#xC0B0; (RA)&#xC740; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xACE8;&#xACA9; myogenesis&#xC758; &#xAC00;&#xC7A5; &#xAC15;&#xB825;&#xD55C; &#xC791;&#xC740; &#xBD84;&#xC790; &#xC99D;&#xAC15;, &#xBB34;&#xB300; &#xBC0F; &#xAE30;&#xB2A5;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xB418;&#xC9C0; &#xC544;&#xC9C1; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xD574;&#xBA85;. &#xB610;&#xD55C;, &#xB2E4;&#xB978; &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xC790;&#xADF9; &#xD558;&#xAC70;&#xB098; &#xC5B5;&#xC81C; myogenesis RA&#xC758; &#xAD50;&#xCC28;&#xB85C; (Wnt &#xBC0F; BMP4, &#xB4F1; &#xAC01;&#xAC01;)&#xB294; &#xC54C; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;,&#xC774; &#xC5F0;&#xAD6C;&#xC758; &#xBAA9;&#xC801;&#xC740;&#xB294; RA &#xACE8;&#xACA9; myogenesis &#xAC15;&#xD654; &#xD558; &#xACE0; Wnt &#xBC0F; BMP4 &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; &#xBD84;&#xC790; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xB3D9;&#xC548; P19 &#xB610;&#xB294; &#xB9C8;&#xC6B0;&#xC2A4; &#xBC30;&#xC544; &#xC904;&#xAE30; (ES&#xC758;) &#xC138;&#xD3EC; &#xBD84;&#xD654; &#xC2E0;&#xD638;.

Retinoic &#xC0B0; &#xACE8;&#xACA9; &#xADFC;&#xC721; &#xC2DC; &#xC870; &#xD615;&#xC131;&#xC744; &#xAC15;&#xD654; &#xD558; &#xACE0; &#xBF08; &#xC804;&#xCCB4;&#xC801; &#xB2E8;&#xBC31;&#xC9C8; 4&#xC5D0; &#xC758;&#xD574; &#xC800;&#xD574; &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xBD80;&#xC815;&#xC801;&#xC778; &#xBCA0;&#xD0C0;-catenin &#xBB34;&#xC2DC;.

Diferenciaci&#xF3;n de las c&#xE9;lulas madre entendimiento es esencial para el futuro dise&#xF1;o de terapias celulares. Mientras que el &#xE1;cido retinoico (RA) es la mol&#xE9;cula peque&#xF1;a m&#xE1;s potente potenciador de miog&#xE9;nesis esquel&#xE9;tico en las c&#xE9;lulas madre, la etapa y el mecanismo de su funci&#xF3;n no ha sido todav&#xED;a dilucidado. Adem&#xE1;s, la intersecci&#xF3;n de la AR con otras v&#xED;as de se&#xF1;alizaci&#xF3;n que estimulan o inhiben miog&#xE9;nesis (como Wnt y BMP4, respectivamente) es desconocida. As&#xED;, el objetivo de este estudio es examinar los mecanismos moleculares por los que la AR aumenta miog&#xE9;nesis esquel&#xE9;tica e interact&#xFA;a con la se&#xF1;alizaci&#xF3;n de Wnt y BMP4 durante el P19 o el rat&#xF3;n madre embrionarias (ES) la diferenciaci&#xF3;n celular.

&#xC1;cido retinoico aumenta la formaci&#xF3;n del esqueleto progenitoras musculares y evita la inhibici&#xF3;n de la prote&#xED;na morfogen&#xE9;tica &#xF3;sea 4, pero no dominante negativo de beta-catenina

Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates.

Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut.

Given that the lateral plate mesoderm (LPM) gives rise to the cardiovascular system, identifying the cascade of signalling events that subdivides the LPM into distinct regions during development is an important question. Retinoic acid (RA) is known to be necessary for establishing the expression boundaries of important transcription factors that demarcate distinct regions along the anterior posterior axis of the LPM. Here, we demonstrate that fibroblast growth factor (Fgf) signalling is also necessary for regulating the expression domains of the same transcription factors (nkx2.5, foxf1, hand1 and sall3) by restricting the RA responsive LPM domains. When Fgf signalling is inhibited in neurula stage embryos, the more posterior LPM expression domains are lost, while the more anterior domains are extended further posterior. The domain changes are maintained throughout development as Fgf inhibition results in similar domain changes in late stage embryos. We also demonstrate that Fgf signalling is necessary for both the initiation of heart specification, and for maintaining heart specification until overt differentiation occurs. Fgf signalling is also necessary to restrict vascular patterning and create a vascular free domain in the posterior end of the LPM that correlates with the expression of hand1. Finally, we show cross talk between the RA and Fgf signalling pathways in the patterning of the LPM. We suggest that this tissue wide patterning event, active during the neurula stage, is an initial step in regional specification of the LPM, and this process is an essential early event in LPM patterning.

Fgf is required to regulate anterior-posterior patterning in the Xenopus lateral plate mesoderm.

The lung and thyroid are derived from the anterior endoderm. Retinoic acid and Fgf signalling are known to be essential for development of the lung in mouse but little is known on how the lung and thyroid are specified in Xenopus.

Retinoic acid is a key regulatory switch determining the difference between lung and thyroid fates in Xenopus laevis.

Retinoic acid signaling is essential for many aspects of early development in vertebrates. To control the levels of signaling, several retinoic acid target genes have been identified that act to suppress retinoic acid signaling in a negative feedback loop. The nuclear protein Ski has been extensively studied for its ability to suppress transforming growth factor-beta (TGF-β) signaling but has also been implicated in the repression of retinoic acid signaling.

Thomas A. Drysdale

Children's Health Research Institute,
Department of Paediatrics

University of Western Ontario

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Thomas A. Drysdale

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Children's Health Research Institute,Department of Paediatrics

University of Western Ontario

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Children's Health Research Institute,
Department of Paediatrics