Germ Plasm Aggregation and Compaction in the Early Zebrafish Embryo

描述(申请人提供)：斑马鱼胚胎中的种质运动和早期肌球蛋白运动。动物发育中最早的细胞命运决定之一是确定原始生殖细胞(PGCs)的分化，这是通过诱导性细胞-细胞相互作用(例如哺乳动物)或预成型(例如硬骨鱼、果蝇、线虫)发生的。预成性(母系遗传)种质是由核糖核粒(RNPs)、蛋白质组成的一种特殊结构，通常与细胞骨架成分有关。斑马鱼模型是研究早期胚胎发育过程中种质运动的理想模型。这些透明的胚胎很容易通过药物、吗啡和RNA注射进行操作。最近，我们实验室的成员改进了一种通过在体外成熟的卵母细胞中注射试剂来操纵作用于胚胎的母体因子的方案，这使得在受精之前和之后立即操纵成为可能。我的初步数据表明，在皱纹形成之前和期间，在种质RNPs的多聚体中存在着磷酸肌球蛋白，这些RNPs存在于F-肌动蛋白上。我们提出了一种机制，通过肌球蛋白II驱动的F-肌动蛋白滑动聚集种质颗粒，并将聚集体移动到前两个形成皱纹的地方。后来，在皱纹成熟过程中，种质RNPs继续进行多聚体，直到它们在这些皱纹的远端形成致密的团块。母系效应致命突变体AURA没有野生型胚胎中看到的同心肌动蛋白环，也不能正确聚集种质RNPs。这表明编码Mid1ip1L的蛋白质AURA参与了种质早期运动的细胞骨架动力学。我计划通过表征种质聚集和在肌动蛋白细丝上的招募来解决这些假设，并确定Mid1ip1L在早期细胞骨架动力学中的作用。提出的GP RNP运动假说将提供一种新的机制，通过肌球蛋白马达对肌动蛋白细胞骨架网络的作用来移动这些细胞决定因素。所研究的机制将有助于理解PGC决定因素的正确运动，这些因素与细胞干细胞、生殖、癌症发生和多能性有关。

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.