Germ Plasm Aggregation and Compaction in the Early Zebrafish Embryo

描述（由申请人提供）：斑马鱼胚胎的种质运动和早期肌动蛋白运动。动物发育中最早的细胞命运决定之一是决定原始生殖细胞（PGCs）的分化，这是通过诱导细胞间相互作用（如哺乳动物）或预形成（如硬骨鱼、果蝇、秀丽隐杆线虫）发生的。预形成（母系遗传）胚质是一种由核糖核酸颗粒（RNPs）、蛋白质组成的特殊结构，通常与细胞骨架成分相关。斑马鱼模型是研究早期胚胎发生过程中种质运动的理想模型。透明的胚胎很容易使用药物，以及morpholino和RNA注射来操纵。最近，我们实验室的成员已经完善了一项通过将试剂注射到体外成熟的卵母细胞中来操纵胚胎中母体因子的方案，这为受精前和受精后立即操纵提供了可能性。我的初步数据表明，在沟形成之前和形成过程中，磷脂凝蛋白存在于种质RNPs中，这些RNPs位于F-肌动蛋白上。我们提出了一种机制，通过肌凝蛋白ii驱动的f -肌动蛋白滑动聚集质粒，并将聚集物移动到前两个形成的沟中。后来，在沟成熟过程中，种质RNPs继续进行多聚，直到它们在这些沟的远端形成致密的团块。母体效应致死突变体aura没有野生型胚胎中的同心圆肌动蛋白环，也不能正确聚集胚质RNPs。这表明aura蛋白编码Mid1ip1L参与了胚质早期运动的细胞骨架动力学。我计划通过在犁沟开始前和犁沟开始时表征肌动蛋白丝上的种质聚集和招募，并确定Mid1ip1L在早期细胞骨架动力学中的作用来解决这些假设。提出的GP RNP运动假说将为这些细胞决定因素通过肌凝蛋白马达对肌动蛋白细胞骨架网络的作用而运动的新机制提供细节。研究的机制将有助于理解pgc决定因子的正确运动，与细胞干细胞、生殖、致癌和多能性有关。

DESCRIPTION (provided by applicant): Germ plasm movement and early acto-myosin movement in the Zebrafish embryo. One of the earliest cell-fate decisions in animal development is the determination of primordial germ cell (PGCs) differentiation, which occurs via inductive cell-cell interactions (e.g. mammals) or preformation (e.g. teleosts, Drosophila, C. elegans). Preformative (maternally inherited) germ plasm is a specialized structure made up of ribonucleoparticles (RNPs), proteins and often associated with cytoskeletal components. The zebrafish model is excellent for studying the movement of germ plasm in early embryogenesis. The transparent embryos are easily manipulated using drugs, as well as morpholino and RNA injections. Recently, members of our laboratory have refined a protocol for manipulation of maternal factors acting in the embryo by injection of reagents into oocytes undergoing in vitro maturation, which has opened the possibility of manipulation prior to and immediately after fertilization. My preliminary data suggest that phosphomyosin is present in germ plasm RNPs during their multimerization prior to and during furrow formation, and that these RNPs reside on F- actin. We propose a mechanism by which myosin II-driven F-actin sliding aggregates germ plasm particles and moves the aggregates into the first two forming furrows. Later, during furrow maturation, germ plasm RNPs continue to undergo multimerization until they form compact masses at the distal ends of these furrows. A maternal effect lethal mutant, aura, does not have the concentric actin rings seen in wild type embryos and do not properly aggregate germ plasm RNPs. This suggests that the protein aura codes for, Mid1ip1L, is involved in the cytoskeletal dynamics of germ plasm early movement. I plan to address these hypotheses by characterizing germ plasm aggregation and recruitment on actin filaments prior to and at furrow initiation, and determining the role Mid1ip1L plays in early cytoskeletal dynamics. The proposed hypothesis of GP RNP movement will provide details on a novel mechanism for the movement of such cellular determinants via the action of a myosin motor on an actin cytoskeletal network. The mechanisms studied will lead to understanding the proper movement of PGC-determining factors, relating to cell stemness, reproduction, carcinogenesis and pluripotency.