Specificaton and Patterning of the Animal-Vegetal Axis

项目概述PI感兴趣的是调节海胆胚胎动植物(A/V)轴规范的母体机制，以及阐明这种母体信息如何用于生殖层分离。沿着A/V轴看到的最早的分子不对称之一是B-连环蛋白进入植物细胞核。核B-连环蛋白是典型的Wnt信号的转导，对于内胚层的确定和沿A/V轴的模式形成都是必不可少的。阐明植物细胞中规范的Wnt信号是如何被激活的，将有助于深入了解指定这一轴的机制。在前一次授精期间所做的工作表明，在受精后，杂乱(Dsh)蛋白变得定位于植物的囊泡皮质结构。DSH与这些膜小泡相互作用，然后调节B-连环蛋白的核进入和规范的Wnt信号的激活。B-连环蛋白在植物细胞中的早期核靶向是信号分子SpWnt8。前一个授权期的结果表明，SpWnt8对于初级间充质分化和内胚层的持续规范是必需的。这些数据表明，SpWnt8在植物细胞中具有早期维持核B-catenin水平的功能，在胚泡晚期具有调节内胚层基因表达和原肠形成的晚期功能。为了进一步了解Dsh和SpWnt8在海胆胚胎中的调节和功能，我们将追求三个特定的目标：1)确定在早期胚胎中调节Dsh活性的分子机制。这些研究将揭示海胆胚胎植物细胞中选择性激活经典Wnt信号的机制，并有可能揭示动植物轴的分子基础；2)从功能上分析关键平面细胞极性通路(PCP)基因在动植物轴模式中的作用。这些研究将揭示通过这种非规范的Wnt途径的信号是否调节早期胚胎的内胚层形成和原肠形成；3)确定SpWnt8激活的细胞内途径来调节内胚层细胞的命运。这些研究将揭示SpWnt8是否可以通过规范和非规范的Wnt通路发出信号，并进一步揭示SpWnt8激活的特定PCP途径。这些研究将增加我们对海胆A/V轴如何指定和模式的理解，并可能为双边动物A/V轴的进化提供洞察力。这些研究还将包括博士后、研究生和本科生，这些学生来自科学界代表性较低的群体。这项工作的结果也将被传达给PI发育生物学课程的学生。

Project SummaryThe PI is interested in the maternal mechanisms that regulate the specification of the animal-vegetal (A/V) axis in the sea urchin embryo, and in elucidating how this maternal information is used in germ layer segregation. One of the first molecular asymmetries seen along the A/V axis is the entry of B-catenin into vegetal cell nuclei. Nuclear B-catenin, which is the transducer of the canonical Wnt signal, is essential for specification of endomesoderm, and also for pattern formation along the A/V axis. Elucidating how canonical Wnt signaling is activated in vegetal cells will provide insight into the mechanisms that specify this axis. Work done during the previous granting period has shown that the Dishevelled (Dsh) protein becomes localized to vegetal vesicular cortical structures following fertilization. Dsh interaction with these membrane vesicles then regulates the nuclear entry of B-catenin and activation of canonical Wnt signaling. An early nuclear target of B-catenin in vegetal cells is the signaling molecule SpWnt8. Results from the previous granting period have shown that SpWnt8 is required for primary mesenchyme differentiation and the continued specification of the endomesoderm. These data suggests that SpWnt8 has an early function to maintain nuclear B-catenin levels in vegetal cells, and a late function at the late blastula stage to regulate endomesoderm gene expression and gastrulation. The late function of SpWnt8 appears to require a non-canonical Wnt signaling pathway that also involves Dsh.To further understand Dsh and SpWnt8 regulation and function in the sea urchin embryo, three specific aims will be pursued: 1) To identify molecular mechanisms that regulate Dsh activity in early embryos. These studies will reveal the mechanisms that selectively activate canonical Wnt signaling in vegetal cells of the sea urchin embryo, and have the potential to reveal the molecular basis for the animal-vegetal axis; 2) To functionally analyze the role of key planar cell polarity pathway (PCP) genes animal-vegetal axis patterning. These studies will reveal if signaling through this non-canonical Wnt pathway regulates endomesoderm formation and gastrulation in the early embryo; 3) To determine the intracellular pathways activated by SpWnt8 to regulate endomesodermal cell fates. These studies will reveal if SpWnt8 can signal via the canonical and non-canonical Wnt pathways, and further, will reveal the specific PCP pathway activated by SpWnt8.Together, these studies will increase our understanding of how the sea urchin A/V axis is specified and patterned and may provide insight into the evolution of the A/V axis in bilaterians. These studies will also include postdocs, graduate students and undergraduate students from groups underrepresented in science. Results from the work will also be communicated to students enrolled in the PI's Developmental Biology classes.