Centrosomal mRNAs in Asymmetric Cell Division

细胞命运在发育过程中由两种机制决定：细胞-细胞信号传导和模式分子的不对称遗传。在这两者中，人们对细胞不对称分裂的了解要少得多。P.I.建议进一步探索在蜗牛Ilyanassa obsoleta胚胎中发现的不对称细胞分裂的新机制，其中图案基因mrna在有丝分裂间期定位于中心体，然后在随后的分裂中不对称遗传。第一个目标是进行原位杂交筛选，以在根据各种标准选择的大约2500个Ilyanassa mrna中找到定位模式。本实验将显示中心体定位的mrna的比例和功能类别，并确定mrna不对称分离的细胞分裂集。该筛选还将确定候选的细胞命运规范基因。第二个目标是破坏局部分子的功能，从而剖析胚胎发育过程的要求。迄今为止观察到的定位模式强烈表明，中心体mrna的遗传对模式很重要。这些实验将证明该机制的功能意义，增加对早期Ilyanassa胚胎中基本模式事件的理解，并作为该系统中基因破坏新开发方法的试验场。最初的研究将集中于在试点筛选中发现的Ilyanassa Nanos蛋白，随后的工作将研究其他候选细胞命运规范基因。智力价值：这些实验的结果将极大地增强我们对不对称细胞分裂新机制的理解，特别是关于它在发育中的功能作用。这种现象位于几个领域的交叉点，为解决它而提出的实验本质上是跨学科的。这项研究将使用发育遗传学、经典胚胎学、细胞生物学、比较生物学和基因组学的工具和概念。更广泛的影响：这项工作有望对理解不对称细胞分裂决定细胞命运的组织的发育产生影响，比如神经系统。它还可能影响对农业上重要的软体动物如双壳类动物发育的研究。这项工作将使大量新cDNA序列在公共数据库中沉积，为利用该动物进行寄生虫学、生态毒理学、化学生态学和幼虫生态学的研究提供依据。这项工作还将为本科生提供参与研究的机会，这项工作的结果将用于P.I.他的发育生物学课程。最后，本文提出的实验将有助于完善和验证研究Ilyanassa基因功能的方法，这将从根本上增强该系统的功能。

Project SummaryCell fates are specified during development by two mechanisms: cell-cell signaling and asymmetric inheritance of patterning molecules. Of the two, asymmetric cell division is much less well understood. The P.I. proposes to further explore a novel mechanism of asymmetric cell division discovered in the embryo of the snail Ilyanassa obsoleta, where mRNAs for patterning genes are localized to centrosomes during mitotic interphase, then asymmetrically inherited during the subsequent division.The first objective is to carry out an in situ hybridization screen to find localization patterns among approximately 2500 Ilyanassa mRNAs, chosen by various criteria. This experiment will show the proportion and functional categories of mRNAs that are centrosomally localized, and determine the set of cell divisions where mRNAs are asymmetrically segregated. This screen will also identify candidate cell fate specification genes.The second objective is to disrupt the function of localized molecules and thus dissect the requirements for this process in embryonic development. The localization patterns observed so far strongly suggest that the inheritance of centrosomal mRNAs is important for patterning. These experiments will demonstrate the functional significance of this mechanism, increase understanding of the basic patterning events in the early Ilyanassa embryo, and serve as a proving ground for newly developed methods of gene disruption in this system. Initial studies will focus on the Ilyanassa Nanos protein discovered in the pilot screen, and subsequent work will examine other candidate cell fate specification genes.Intellectual merit: The results of these experiments should dramatically enhance our understanding of a novel mechanism of asymmetric cell division, especially with regard to its functional role in development. The phenomenon lies at the intersection of several fields and the experiments proposed to address it are intrinsically interdisciplinary. This research will use tools and concepts from developmental genetics, classical embryology, cell biology, comparative biology, and genomics.Broader impact: This work is expected to have implications for understanding the development of tissues where asymmetric cell division determines cell fate, like the nervous system. It may also impact studies of development in agriculturally important molluscs like bivalves. The work will result in the deposition of a large number of new cDNA sequences for Ilyanassa obsoleta in public databases, which should aid those who use this animal for studies in parasitology, ecotoxicology, chemical ecology and larval ecology. The work will also provide opportunities to involve undergraduates in research, and results from this work will be used in the P.I.'s Developmental Biology course. Finally, the experiments proposed here will serve to refine and validate methods for studying gene function in Ilyanassa, which will fundamentally enhance the power of this system.