Specificaton and Patterning of the Animal-Vegetal Axis

该项目主要研究海胆胚胎中调节动物-植物（A/V）轴发育的母系机制，以及母系信息如何用于胚层分离。沿A/V轴观察到的第一个分子不对称是b -连环蛋白进入植物细胞核。核b -连环蛋白是典型Wnt信号的换能器，对内胚层的规范和沿A/V轴的模式形成至关重要。阐明典型Wnt信号是如何在植物细胞中被激活的，将有助于深入了解指定该轴的机制。之前的研究表明，disheveled （Dsh）蛋白在受精后定位于植物囊泡皮质结构。Dsh与这些膜泡的相互作用调节b -连环蛋白的核进入和典型Wnt信号的激活。b -连环蛋白在植物细胞中的早期核靶标是信号分子SpWnt8。先前的研究结果表明，SpWnt8是原发性间质分化和内胚层持续分化所必需的。这些数据表明，SpWnt8在植物细胞中具有维持细胞核b -连环蛋白水平的早期功能，而在囊胚后期具有调节内胚层基因表达和原肠形成的晚期功能。SpWnt8的晚期功能似乎需要一个非规范的Wnt信号通路，该通路也涉及Dsh。为了进一步了解Dsh和SpWnt8在海胆胚胎中的调控和功能，我们将从三个方面进行研究：1)确定早期胚胎中Dsh活性的调控分子机制。这些研究将揭示海胆胚胎植物细胞中选择性激活典型Wnt信号的机制，并有可能揭示动物-植物轴的分子基础；2)从功能上分析平面细胞极性通路（PCP）关键基因在动物-植物轴模式中的作用。这些研究将揭示通过这种非规范Wnt通路的信号是否调节早期胚胎的内膜形成和原肠胚形成；3)确定SpWnt8激活的细胞内通路调控内胚层细胞命运。这些研究将揭示SpWnt8是否可以通过典型和非典型Wnt通路发出信号，并进一步揭示SpWnt8激活的特定PCP通路。总之，这些研究将增加我们对海胆A/V轴是如何指定和图案的理解，并可能为双边动物A/V轴的进化提供见解。这些研究还将包括来自科学领域代表性不足群体的博士后、研究生和本科生。这项工作的结果也将传达给参加PI的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.