Specification and Patterning of the Animal-Vegetal Axis

0110532Wikramanayake 早期海胆胚胎中核β-连环蛋白的消耗会影响动物-植物 (A/V) 轴上多个细胞谱系的命运。众所周知，微粒形成了中内胚层的早期信号传导中心，需要核β-连环蛋白才能发挥其诱导功能。 veg2 谱系中也需要 β-连环蛋白信号传导，以便这些细胞对微信号做出反应并被指定为中内胚层。识别β-连环蛋白的转录靶点并阐明其功能将是定义​​中内胚层特异性分子基础的关键步骤。该提案将重点关注 SUWnt8，这是一种核β-连环蛋白敏感的 Wnt 基因，该基因在微体中被激活，与 β-连环蛋白进入这些细胞的细胞核同时发生，并且在 β-连环蛋白进入该细胞层的细胞核后不久在 veg2 层中被激活。提出的四个具体目标将定义 SUWnt8 在这些细胞谱系中的功能，并将进一步了解海胆胚胎中内胚层的规范。这些目标是： (1) 确定细胞相互作用在调节中内胚层区域早期 SUWnt8 表达中的作用。这将有助于深入了解细胞自主和非细胞自主机制在早期中内胚层规范中的相对作用。 (2) 通过使用吗啉代反义寡核苷酸介导的翻译抑制来阻断其活性，阐明 SUWnt8 在微粒和 veg2 层中的作用，以确定 SUWnt8 如何参与早期中内胚层规范。此外，还将确定 SUWnt8 在维持植物细胞核 β-连环蛋白水平和抑制植物细胞动物化转录因子活性方面的可能作用。 (3) 虽然正常中内胚层规范需要核β-连环蛋白，但有证据表明，在缺乏可检测到的核β-连环蛋白的情况下，微粒可以在动物半卵裂球中诱导中内胚层。将确定 SUWnt8 是否可以通过 Disheveled 蛋白通过平面细胞极性途径发出信号，从而与 β-连环蛋白途径并行参与中内胚层规范。 (4)SUWnt8独特的表达模式使其成为研究中内胚层规范转录基础的有力试剂。将鉴定介导微粒和 veg2 层中 SUWnt8 表达的反式作用因子。 总之，这些研究将进一步加深我们对海胆 A/V 轴如何指定和模式化的理解，并可能提供对两侧对称动物 A/V 轴进化的深入了解。

0110532WikramanayakeDepletion of nuclear beta-catenin in early sea urchin embryos affects thefates of multiple cell lineages along the animal-vegetal (A/V) axis. It isknown that the micromeres, which form the early signaling center thatspecifies mesendoderm, require nuclear beta-catenin to become competent tocarry out their inductive functions. Beta-catenin signaling is alsorequired in the veg2 lineage for these cells to respond to micromeresignals and to become specified as mesendoderm. Identifyingtranscriptional targets of beta-catenin and elucidating their functionswould be a critical step in defining the molecular basis of mesendodermspecification. This proposal will focus on SUWnt8, a nuclearbeta-catenin-sensitive Wnt gene that is activated in the micromerescoincident with the entry of beta-catenin into the nuclei of these cellsand in the veg2 tier shortly after transit of beta-catenin into the nucleiof this cell tier. Four proposed specific aims will define the function ofSUWnt8 in these cell lineages and will further the understanding ofmesendoderm specification in the sea urchin embryo. These aims are: (1) Todetermine the role of cell interactions in regulating early SUWnt8expression in the mesendoderm domain. This will provide insight into therelative roles of cell autonomous and non cell-autonomous mechanisms inearly mesendoderm specification. (2) To determine how SUWnt8 participatesin early mesendoderm specification by elucidating its role in themicromeres and the veg2 tier by blocking its activity using morpholinoantisense oligonucleotide-mediated translational inhibition. Additionally,a possible role for SUWnt8 in maintaining nuclear beta-catenin levels invegetal cells and in suppressing the activity of animalizing transcriptionfactors in vegetal cells will be determined. (3) While nuclearbeta-catenin is required for normal mesendoderm specification, there isevidence that micromeres can induce mesendoderm in animal half blastomeresin the absence of detectable nuclear beta-catenin. It will be determinedif SUWnt8 can signal through the planar cell polarity pathway via theDisheveled protein to participate in mesendoderm specification in parallelwith the beta-catenin pathway. (4) The unique expression pattern of SUWnt8makes it a powerful reagent for studying the transcriptional basis ofmesendoderm specification. The trans-acting factors that mediate SUWnt8expression in the micromeres and the veg2 tier will be identified.Together, these studies will further our understanding of how the seaurchin A/V axis is specified and patterned and may provide insight intothe evolution of the A/V axis in bilaterians.