The Physiological Basis of Polarity Formation in Pelvetia

发育的某些方面不能仅仅通过研究差异基因活性的时间进程和一个基因的产物对级联中下一个基因的顺序影响来理解。胚胎中空间模式的出现，在某种程度上肯定是由外部因素的不对称影响引起的。极化的影响可能是附属细胞在卵巢中的不对称位置，如昆虫毛囊的哺育细胞。外部不对称性的结果是卵子内蛋白质和信使核糖核酸的非随机分布，因此这些分子可能在第一次分裂时差异地分布到子细胞。这一主题在开发的后期阶段会重演。上述初级偏振事件的细节很难研究。涉及的牢房往往很小或难以接近。即使重要试剂的身份是已知的，也可能无法控制影响极性的外部梯度。长期以来，褐藻Fucus和Pelvetia的卵一直被用作这些研究的模型系统，最近的工作导致了藻卵和其他发育系统之间机制的显著趋同。最初的无极合子在一段时间内将光梯度转化为发育梯度，最终在一个位置萌发，表达类根-叶状体轴。第一次细胞分裂垂直于这条轴，结果形成了两个不同命运的细胞。本实验室最近的研究表明，光诱导的细胞质Ca~(2+)梯度的形成以小时为单位进行萌发，未来根状茎一侧的Ca~(2+)浓度较高。CGMP也有强制性的增加，有证据表明光感受器可能是一种视蛋白样蛋白。这项拟议的研究旨在利用检测细胞质钙离子梯度的能力来严格测试梯度指导轴形成的假设。例如，当细胞被外部梯度而不是光极化时，梯度是否以预测的方式形成？当通过反转光线方向来反转施加的极性时，渐变会以预期的方式重新形成吗？钙的来源是细胞外的，还是从细胞内的储备库中释放出来的？Aequorin将被用作钙离子传感器，以提高时间分辨率。上述实验中使用的方法时间分辨率较差。Aequorin的初步结果表明，过程的重要细节因此被遗漏了。设计了一种新的方法，可以测量具有良好的时间分辨率和空间分辨率的赤道光。自参比离子特定电极将被用来确定极化过程中离子(特别是钙离子，但也包括H+和K+)的局部净流入和流出。关于钙离子通量如何相对于钙离子梯度的形成而变化的细节，可能使区分光如何导致梯度形成的不同可能性成为可能。光感受器是视蛋白样蛋白的假设将受到挑战。视网膜对失明细胞恢复光敏性的能力将被测试。提取的膜蛋白将用多种抗视蛋白抗体进行筛选。放射性视网膜将被用于标记推定的视蛋白。我们将用从视蛋白保守区设计的寡核苷酸来筛选Fucus基因文库，以期通过聚合酶链式反应证实视蛋白基因的存在。

There are aspects of development that cannot be understood solely by the study of the temporal progression of differential gene activity and the sequential effects of one gene's product on the next gene in a cascade. The emergence of spatial pattern in the embryo must, at some point, arise from the asymmetric influence of an external agent. The polarizing influences may be the asymmetrical location of accessory cells in the ovary, such as the nurse cells of the insect follicle. The result of external asymmetries is the non-random distribution of proteins and mRNA within the egg so that these molecules may be differentially distributed to the daughter cells at first division. This motif is replayed at later stages of development. The details of the primary polarization events described above are difficult to study. The cells involved are often small or inaccessible. It may not be possible to control the external gradients that influence polarity, even it the identity of the important agents are known. The eggs of the brown algae, Fucus and Pelvetia, have long been used as a model system for these studies, and recent work has led to a remarkable convergence of mechanisms between the algal eggs and other developmental systems. The initially apolar zygotes convert a light gradient into a developmental gradient over a period of hours, eventually germinating at one locus to express the rhizoid-thallus axis. First cell division is perpendicular to this axis with the result that two cells with different fates are formed. Recent studies in this laboratory have demonstrated the formation of light-induced cytoplasmic Ca2+ gradients that proceed germination by hours, with high Ca2+ on the future rhizoidal side. There is also an obligatory increase in cGMP and there is evidence that the photoreceptor may be an opsin-like protein. The proposed research aims to exploit the ability to detect cytoplasmic Ca2+ gradients to rigorously test the hypothesis that the gradient directs the formation of an axis. For example, does a gradient form in the predicted way when the cells are polarized by external gradients other than light? Does the gradient re-form in the expected way when an imposed polarity is reversed by reversing the light direction? Is the source of the Ca2+ extracellular or is some released from intracellular stores? Aequorin will be used as a Ca2+ sensor to give improved temporal resolution. The method used in the experiments described above has poor temporal resolution. Preliminary results with aequorin indicate that important details of process are thus missed. A new method has been devised to permit the measurement of aequorin light with excellent temporal resolution and a degree of spatial resolution. Self-referencing ion specific electrodes will be used to determine the localized net influx and efflux of ions (especially Ca2+, but also H+ and K+) during the polarization process. The details of how Ca2+ fluxes change with respect to the formation of a Ca2+ gradient may make it possible to distinguish among alternate possibilities of how light leads to formation of gradients. The hypothesis that the photoreceptor is an opsin-like protein will be challenged. The ability of retinal to restore photosensitivity to blinded cells will be tested. Extracted membrane proteins will be screened with a variety of anti-opsin antibodies. Radioactive retinal will be employed in an effort to label the putative opsin. A Fucus cDNA library will be screened with oligonucleotides designed from conserved regions of opsins in an effort to demonstrate the existence of an opsin gene via PCR.