Establishment of the Growth Axis and Alignment of the Division Plane in Pelvetia Zygotes

胚胎轴的建立是真核生物发育的基础，但由于胚胎保存在母体组织中，难以分离，因此对植物的这一过程知之甚少。相比之下，褐藻（Fucus sp.和Pelvetia sp.）的受精卵很容易从海水中获取，长期以来一直作为研究极性建立的模式生物。在过去的9年里，Kropf实验室一直在研究压缩木受精卵的细胞生物学和极化生理学，主要关注细胞质离子和细胞骨架的作用。基于这项研究和其他实验室的研究，克罗夫夫实验室制定了一个工作模型，以考虑生长轴的建立和划分平面的精确方向。简而言之，该模型假设生长轴的根状极（根状体-块状体）由一块皮质f -肌动蛋白标记，分裂平面由核膜上中心体的方向决定。假设精子进入通过在精子进入位点引起f -肌动蛋白贴片的组装和将中心粒引入卵细胞质来诱导两个轴。在发育的最初几个小时里，f -肌动蛋白贴片以及生长轴可以根据环境因素重新定位。中心体轴不响应环境线索，因此两个轴相互独立地发育。PI推测，在根状极的f -肌动蛋白贴片确定了分泌的靶点，分子的极性分泌局部进入根状质膜和细胞壁放大了新生生长轴。在根状极开始生长后，在第一次有丝分裂之前，中心体轴旋转成与生长轴平行的直线。纺锤极由对齐的中心体形成，细胞质分裂将纺锤体一分为二，确保分裂平面与生长轴横向。拟议的研究将测试这一工作模型的突出方面。主要目的是：1)确定精子进入是否诱导初始极性2)证明一个f -肌动蛋白斑块标志着轴的根状极3)研究极性分泌的机制4)分析微管（MTs）在中心体旋转排列中的作用5)阐明质膜-细胞壁粘附在旋转排列中的作用6)通过胞质H+活性研究细胞骨架排列的调节首次全面了解了细胞骨架在岩藻样受精卵极性建立和表达中的作用，并将开启有关极性分泌和H+调控细胞骨架的重要研究。本研究也将为今后高等植物胚胎极性建立的研究提供一个范例。

Establishment of embryonic axes is fundamental to the development of eukaryotic organisms, yet very little is known about this process in plants because the embryo is retained inside maternal tissues and is difficult to isolate. In contrast, zygotes of the brown algae, Fucus sp. and Pelvetia sp., are easily harvested from seawater and have long served as model organisms for investigation of polarity establishment. For the past nine years the Kropf lab has been studying the cell biology and physiology of polarization in P. compressa zygotes focusing primarily on the roles of cytosolic ions and the cytoskeleton. Based upon this research, and that from other laboratories, the Kropf lab has formulated a working model to account for establishment of the growth axis and the precise orientation of the division plane. Briefly, this model postulates that the rhizoid pole of the growth (rhizoid-thallus) axis is marked by a patch of cortical F-actin and that the division plane is determined by the orientation of centrosomes on the nuclear envelope. Sperm entry is postulated to induce both axes by causing assembly of an F-actin patch at the sperm entry site and by introducing centrioles into the egg cytoplasm. Over the first few hours of development, the F-actin patch, and hence the growth axis, can be repositioned in response to environmental cues. The centrosomal axis does not respond to environmental cues and therefore the two axes develop independently of one another. The PI speculates that the F-actin patch at the rhizoid pole defines a target site for secretion, and that polar secretion of molecules locally into the rhizoid plasma membrane and cell wall amplifies the nascent growth axis. After growth begins at the rhizoid pole, and just prior to first mitosis, the centrosomal axis rotates into parallel alignment with the growth axis. The spindle poles form from the aligned centrosomes and cytokinesis bisects the spindle, ensuring that the division plane is transverse to the growth axis.The proposed research will test the salient aspects of this working model. The primary objectives are to:1) determine whether sperm entry induces an initial polarity2) demonstrate that a patch of F-actin marks the rhizoid pole of the axis3) investigate the mechanism of polar secretion4) analyze the role of microtubules (MTs) in rotational alignment of the centrosomes5) clarify the role of plasma membrane-cell wall adhesions in rotational alignment6) investigate regulation of the cytoskeletal arrays by cytosolic H+ activityConfirmation of our working model will provide the first comprehensive picture of the role of the cytoskeleton in polarity establishment and expression in fucoid zygotes, and will initiate important studies on polar secretion and cytoskeletal regulation by H+. This study will also provide a paradigm for future investigations of polarity establishment in higher plant embryos.