Maternal Control of Pattern Formation at the Ends of the Drosophila Embryo

项目概述看似简单的受精卵产生复杂的多细胞生物体的机制是一个自亚里士多德时代以来就一直思考的基本感兴趣的话题。果蝇早期发育研究的一个主要发现是，胚胎中花纹的产生依赖于卵泡中先前建立的花纹，在卵泡中形成卵子。母蝇为发育中的卵子提供了一组局部决定因素，这些决定因素稍后将指导胚胎自身基因的空间特异性表达。在由局部母系决定因素指定的结构中，果蝇胚胎中最末端的结构是位于前部的梯状结构和位于后部的端部。末端的形成需要跨膜受体酪氨酸激酶躯干的激活，该躯干分布在早期胚胎的整个膜上。这种激活必须特别发生在胚胎的末端。躯干的配体被认为是躯干蛋白，它被分泌到充满液体的卵黄周空间，该空间位于胚胎膜和蛋壳内表面，即卵黄膜之间。Trunk对躯干的空间受限激活被认为是由Torsolike基因的产物控制的。在卵子发生过程中，Torsolike在发育中的卵母细胞的前端和后端的两个特殊的极型毛囊细胞群中表达。我们的实验室发现，龟板位于蛋壳的前端和后端。这些观察表明了一种机制，即一种不活跃的前体形式的Trunk被均匀地分泌到卵周隙中，并在卵子的末端被加工成活性配体，然后在那里它结合并激活躯干受体。躯干激活需要Torsolike在一个尚未阐明的过程中发挥作用。这项工作的目的是确定Trunk被激活的分子机制，并阐明Torsolike蛋白在这一过程中所起的作用。具体目标是：(1)确定Trunk是否从非活性前体加工成活性配体。(2)通过与Torsolike相互作用的能力，以及通过对影响末端规范的新突变的表征，确定参与Trunk配体形成的其他效应分子。(3)确定在蛋壳卵黄膜上介导其定位的托螺体决定因素。智力价值：除了对早期发育模式形成的理解做出贡献外，这些实验还具有超出果蝇早期发育背景的意义。在许多生理和发育过程中，包括轴突寻路、细胞迁移、细胞分化和增殖在内的多种细胞/细胞通讯通路都需要通过受体酪氨酸激酶进行信号转导。果蝇躯干受体在胚胎模式形成中的作用是一个特别引人注目的范例，因为它的激活在空间和时间上都有精细的调节。通过其配体控制躯干的激活可能会提供重要的见解，适用于各种其他具有智力和医学价值的配体/受体的相互作用。拟议活动产生的更广泛影响：(1)发现、理解、教学、培训和学习。果蝇胚胎前后极的母性控制是发育生物学本科教学中使用的一种教科书范式。这里描述的实验寻求对这一过程的更深层次的理解。此外，拟议的调查为德克萨斯大学本科生的实验贡献提供了许多机会，这将有助于在研究环境中对这些学生进行“动手”培训。有兴趣的本科生将被鼓励参与这个项目。这些学生将有机会在地方和国家科学会议上展示他们的工作。由研究生研究员和教员助理组成的研究团队也将从事本科教学工作。(2)代表性不足的群体的参与。德克萨斯大学录取了大量来自历史上代表性不足的群体的学生。来自这些小组的有兴趣的学生将有机会接受与拟议研究有关的实验室研究的动手培训。(3)成果传播。所获得的成果将发表在科学文献上，并在科学会议上报告。此外，拟议调查的部分涉及产生可能对其他调查人员有用的试剂。这些文件将免费分发给任何提出要求的人。

Project SummaryThe mechanisms through which the seemingly simple fertilized egg generates a complex multicellular organism is a topic of fundamental interest that has been pondered since the time of Aristotle. A major insight that has emerged from studies of early development in the fruitfly Drosophila melanogaster is that the generation of pattern in the embryo is dependent upon the prior establishment of pattern in the ovarian follicle in which the egg is formed. The mother fly provisions the developing egg with a set of localized determinants that will later direct the spatially- specific expression of the embryo's own genes. Among the structures that are specified by localized maternal determinants are the most terminally-positioned structures of the Drosophila embryo, the acron at the anterior and the telson at the posterior. Formation of the termini requires activation of the transmembrane receptor tyrosine kinase Torso, which is distributed throughout the membrane of the early embryo. This activation must occur specifically at the ends of the embryo. The ligand for Torso is thought to be the Trunk protein, which is secreted into the fluid-filled perivitelline space that lies between the embryonic membrane and the inner surface of the eggshell, the vitelline membrane. Spatially-restricted activation of Torso by Trunk is believed to be controlled by the product of the gene torsolike. During oogenesis, torsolike is expressed in two specialized groups of polar follicle cells at the anterior and posterior ends of the developing oocyte. Our laboratory has found that Torsolike is localized to the anterior and posterior ends of the eggshell. These observations suggest a mechanism whereby an inactive precursor form of Trunk is secreted uniformly into the perivitelline space and processed into an active ligand specifically at the ends of the egg where it then binds and activates the Torso receptor. Trunk activation requires the function of Torsolike in a process that has not been elucidated. The objectives of this work are to determine the molecular mechanism by which Trunk becomes activated, and to elucidate the role Torsolike protein plays in this process. The specific aims are: (1) To determine whether Trunk is processed from an inactive precursor into an active ligand. (2) To identify other effector molecules involved in the formation of the Trunk ligand by their abilities to interact with Torsolike, and through the characterization of new mutations that affect the specification of the termini. (3 ) To identify the determinants of Torsolike that mediate its localization in the vitelline membrane of the eggshell. Intellectual merit: In addition to their contribution to the understanding of pattern formation in early development these experiments have significance outside of the context of Drosophila early development. Signal transduction through receptor tyrosine kinases is required for a wide variety of cell/cell communication pathways that operate in numerous physiological and developmental contexts, including axonal pathfinding, cell migration, cell differentiation and proliferation. The role of the Drosophila Torso receptor in embryonic patterning is a particularly compelling paradigm because of the exquisite regulation of its activation, both spatially and temporally. The control of Torso activation by its ligand is likely to provide important insights applicable to a variety of other ligand/receptor interactions that are of intellectual, as well as medical interest. Broader impacts resulting from the proposed activity: (1) Discovery, understanding, teaching, training and learning. The maternal control of Drosophila embryonic anterior-posterior polarity is a textbook paradigm that is used in undergraduate teaching of developmental biology. The experiments described herein seek a deeper understanding of this process. Furthermore, the investigations proposed provide numerous opportunities for experimental contributions by undergraduate students at the University of Texas, which will contribute to "hands on" training of those students in a research setting. Interested undergraduate students will be encouraged to participate in this project. These students will have the opportunity to present their work at local and national scientific meetings. Both the graduate student researcher and faculty associates comprising the research team on this proposal will also engage in undergraduate teaching. (2) Participation of underrepresented groups. The University of Texas matriculates a large population of students from historically underrepresented groups. Opportunities for hands on training in laboratory research related to the proposed studies will be available to interested students from these groups. (3) Dissemination of Results. Results obtained will be published in the scientific literature and reported at scientific conferences. Moreover, segments of the proposed investigations involve the generation of reagents likely to be of use to other investigors. These will be disseminated freely to any who request them.