Signaling From the Molluscan D Quadrant Organizer

泥蜗牛Ilyanassa obsoleta长期以来一直是发育机制研究的模型，因为它提供了一个壮观的例子，本地化的细胞质决定因素。 伊利亚那萨有一种独特的细胞分裂模式，称为螺旋分裂，它与其他几个门共享。此外，它在头两次分裂时挤出一个细胞质球-极叶。当去除极叶时，胚胎缺乏背腹轴，具有径向对称性。不仅极叶消融胚胎中缺少极叶的直接物理后代，而且无叶胚胎缺乏已知从其他细胞遗传下来的结构。因此，通过细胞质定位的过程，极叶为胚胎中的一个或多个细胞提供了诱导其他细胞命运的能力。因此，极叶决定因素的本地化指定的组织者的Ilyanassa背腹轴。尽管发现极瓣效应已经有100多年了（Crampton，1896），组织活动定位的分子机制以及组织活动本身的性质才刚刚被发现。Nagy博士已经确定了软体动物组织者的第一个已知成分：有丝分裂原活化蛋白激酶（MAPK）的局部活化。MAPK活化最初在信号传导细胞（3D大单体）中活化。MAPK在所有需要诱导信号进行正常发育的细胞中被二次激活，并且在功能上是第二和第三四个四分之一微粒特化所需的。基于我们的初步数据，她构建了Ilyanassa组织者的建立和功能的以下模型：MAPK是由于遗传极叶决定簇而在信号细胞中自主激活的细胞（3D）。一旦激活，它会触发至少两个信号的释放。第一个信号是尚未确定的，其功能是激活PKC和MAPK的一个子集的微粒。3D中的MAPK还通过调节DPP mRNA翻译激活第二个信号DPP。释放DPP配体的功能是形成第一个四联体微粒，其后代将成为眼睛。这项资助的目的是通过探索激活3D大单体中MAPK的机制以及3D信号向胚胎其余部分传递的机制来测试该模型的每个步骤。更具体地说，我们提出了以下目的：目的1）通过进行一系列消融实验来测定非D象限细胞对3D中MAPK激活的贡献，以确定极叶的遗传是否是必需的和足够的。目的2）通过分析24-细胞胚胎中细胞之间的物理相互作用，来表征3D信号向微粒传递的机制的性质。目的3）通过生物化学抑制已知调节PKC（微粒）和MAPK激活（3D和微粒）的主要途径的组分，来鉴定3D和微粒中MAPK激活的上游因子。目的4）。通过分析极叶切除和MAPK抑制胚胎中的Dpp、tld和pSMAD表达，并采用过表达和敲低功能的方法测定其功能，确定DPP信号传导是否在3D中MAPK下游发挥作用，以形成微粒子的一个子集。Nagy博士在Ilyanassa 3D信号传导的分子基础上的发现使她能够整合世纪的胚胎学操作，将软体动物的组织者理解为分子框架。一旦完成，拟议的实验将显着增加我们的理解，在软体动物的发展诱导信号。 由于软体动物胚胎的细胞分裂和细胞命运与许多不同门的胚胎有着惊人的相似性，因此深入了解Ilyanassa的模式化机制将对我们了解其他软体动物和Lophotrochozoan胚胎产生重大影响。更广泛的影响：本项目促进多层次的探究教育。一些拟议的工作形式的研究生培养的基础;其他方面将形成本科生的研究项目。目前从事该项目的一名研究生正在使用她在该系统中的结果来设计发育生物学的在线教学模块。我们的研究结果将发表在科学期刊上，并定期在地方，国家和国际会议上发表，并在本科课程，研讨会和在线讨论部分进行讨论。

PROJECT SUMMARYThe mud snail Ilyanassa obsoleta has long been a model for the study of developmental mechanisms because it provides a spectacular example of localized cytoplasmic determinants. Ilyanassa has a distinctive pattern of cell division known as spiral cleavage, which it shares with several other phyla. In addition, it extrudes a cytoplasmic bulb - the polar lobe- during its firsttwo cleavages. When the polar lobe is removed, the embryo lacks a dorsal-ventral axis and has radial symmetry. Not only are the direct physical progeny of the polar lobe missing in polar lobe ablated embryos, but lobeless embryos lack structures known to descend from other cells. Thus, through a process of cytoplasmic localization, the polar lobe provides one or more cells in theembryo with the ability to induce the fates other cells. The localization of polar lobe determinants thus specifies the organizer of the Ilyanassa dorsal-ventral axis. Although it has been more than 100 years since the discovery of the effects of the polar lobe (Crampton, 1896), the molecular mechanisms underlying the localization of the organizing activity, as well as the nature of the organizing activity itself, are only just being discovered.Dr. Nagy has identified the first known component of the molluscan organizer: a localized activation of mitogen-activated protein kinase (MAPK). MAPK activation is initially activated in the signaling cell, the 3D macromere. MAPK is secondarily activated in all cells that require the inductive signal for normal development and is functionally required for specification of second and third quartet micromeres. Based on our preliminary data, she has constructed thefollowing model for the establishment and function of the Ilyanassa organizer: MAPK is activated cell autonomously in the signaling cell (3D) as a result of inheriting polar lobe determinants. Once activated, it triggers the release of at least two signals. The first signal is as yet unidentified, and functions to activate PKC and MAPK in a subset of the micromeres. MAPK in 3D also activates a second signal, DPP, by regulating dpp mRNA translation. Release of the DPP ligand functions to pattern the first quartet micromeres, whose progeny willbecome eyes. The aims of this grant are to test each of the steps of this model by probing the mechanisms that activate MAPK in the 3D macromere and the mechanisms by which the 3D signals to the rest of the embryo. More specifically, we propose the following aims:Aim 1) To determine whether the inheritance of the polar lobe is necessary and sufficient to activate MAPK in 3D, by performing a series of ablation experiments to assay the contribution of non-D quadrant cells to MAPK activation in 3D.Aim 2) To characterize the nature of the mechanism by which 3D signals to the micromeres, by analyzing the physical interactions between cells in the 24-cell embryo.Aim 3) To identify factors upstream of MAPK activation in both 3D and the micromeres by biochemically inhibiting components of the major pathways known to regulate PKC (micromeres) and MAPK activation (3D and micromeres).Aim 4). To determine whether DPP signaling functions downstream of MAPK in 3D to pattern a subset of the micromeres, by analyzing Dpp, tld and pSMAD expression in polar lobe ablated and MAPK inhibited embryos and assay their function with methods to over-express and knock-down function.Intellectual Merit: Dr. Nagy's findings on the molecular basis of Ilyanassa 3D signaling puts her in a position to integrate a century of embryological manipulations aimed at understanding the molluscan organizer into a molecular framework. Once completed, the proposed experiments will significantly increase our understanding of inductive signaling in molluscan development. As molluscan embryos share striking similarities of cell division and cell fates with embryos of a number of different phyla, insight into the patterning mechanism of Ilyanassa will have significant impact on our understanding of other molluscan and lophotrochozoan embryos.Broader impact: This project promotes education at multiple levels of inquiry. Some of the proposed work forms the basis of graduate training; other aspects will form undergraduate research projects. One of the graduate students currently working on the project is using her results in this system to design an online teaching module for developmental biology. The results of our research will be published in scientific journals and regularly presented at local, national and international meetings and discussed in undergraduate classes, seminars, and online discussion sections.