Microtuble Organization and Dynamics During Amphibian Oogenesis

两栖动物的卵沿着动物-植物（A-V）轴极化，这是由色素和蛋黄的不对称分布以及减数分裂纺锤体的位置决定的。卵发生过程中形成的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涉及到A-V轴的规范，尽管精确的机制尚不清楚。本实验旨在确定非洲爪蟾卵发生和A-V轴形成过程中微管和微管组织中心的组织和动力学。共聚焦免疫荧光显微镜将用于检查卵发生过程中总微管和乙酰化（非动态）微管的组织。微注射生物素化微管蛋白将用于表征I期卵母细胞、III-IV期A-V轴形成和卵母细胞成熟过程中的微管组装位点和动力学。微注射荧光素标记的微管蛋白和时移共聚焦显微镜将用于检查活的I期卵母细胞和减数分裂纺锤体组装和排列过程中的微管动力学。非洲爪蟾卵母细胞是一个大的球形细胞，在其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，但很重要，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的作用的重要信息。

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.