Microtuble Organization and Dynamics During Amphibian Oogenesis

两栖虫卵沿着动植物(A-V)轴极化，该轴由色素和蛋黄的不对称分布以及减数分裂纺锤体的位置定义。在卵子发生过程中形成的A-V轴在胚胎发育后期胚层的建立中起着重要作用。微管和微管组织中心经常与房室轴的指定有关，尽管确切的机制尚不清楚。本实验旨在确定非洲爪哇卵子发生和A-V轴形成过程中微管和微管组织中心的组织和动态。共聚焦免疫荧光显微镜将被用来检查卵子发生过程中总微管和乙酰化(非动态)微管的组织。显微注射生物素化微管蛋白将被用来研究第I期卵母细胞、第III-IV期A-V轴形成期间和卵母细胞成熟过程中微管组装的位置和动力学。显微注射荧光素标记的微管蛋白和延时共聚焦显微镜将被用来研究活着的I期卵母细胞以及减数分裂纺锤体的组装和排列过程中的微管动力学。非洲爪哇的卵母细胞是一个大的球形细胞，在发育早期表现出细胞质成分的非随机分布。控制这种再分配的机制尚不清楚，但意义重大，因为它是胚胎极化生长的基础。该项目将提供有关微管和微管组织中心在该细胞的动植物轴形成中所起作用的重要信息。

Amphibian eggs are polarized along an animal-vegetal (A-V) axis defined by the asymmetric distribution of pigment and yolk, and by the location of the meiotic spindles. The A-V axis formed during oogenesis plays an important role in the establishment of the germ layers during later embryonic development. Microtubules and microtubule organizing centers have often been implicated in specification of the A-V axis, though the precise mechanism remains unknown. The proposed experiments are designed to determine the organization and dynamics of microtubules and microtubule organizing centers during oogenesis and A-V axis formation in Xenopus laevis. Confocal immunofluorescence microscopy will be used to examine the organization of total and acetylated (non- dynamic) microtubules during oogenesis. Microinjection of biotinylated-tubulin will be used to characterize the microtubule assembly sites and dynamics in stage I oocytes, during A-V axis formation in stages III-IV, and during oocyte maturation. Microinjection of fluorescein-labeled tubulin and time-lapse confocal microscopy will be used to examine microtubule dynamics in living stage I oocytes and during assembly and alignment of the meiotic spindles. %%% The oocyte of Xenopus laevis is a large spherical cell that exhibits a nonrandom distribution of cytoplasmic components early in its development. The mechanism that controls this redistribution is not known, but is significant because it underlies polarized growth of the embryo. This project will provide important information on the roles of microtubules and microtubule organizing centers on the formation of the animal- vegetal axis of this cell.