Mechanisms for Measuring the Nucleocytoplasmic Ratio in Early Embryogenesis

描述(申请人提供)：生物系统的一个基本特性是协调细胞增殖和调节细胞大小。这种协调确保细胞在分裂前生长到最小尺寸，并起到检查点的作用，防止不受限制的增殖。虽然目前还不完全了解细胞如何测量它们的大小，但人们已经意识到，细胞大小的确定与基因组DNA与细胞质体积的比率有关。这表明存在测量核质(N：C)比率的生物学机制，但该机制的组成部分目前尚不清楚。本研究拟以果蝇母体向合子转变的研究为模型，进一步了解N：C比测量的机制。早期胚胎将从母体发育控制到合子发育的转变与N：C比率的测量结合在一起。这一转变导致了胚胎基因表达的全球变化，部分原因是翻译调节机制和染色质修饰机制的协调活动。然而，由于缺乏专门破坏这一过程的突变体，对这种协调背后的机制的准确描述一直受到阻碍。这项拟议的研究将检查最近发现的两个在N：C比率测量方面存在潜在缺陷的果蝇突变体(dfmr1/CAPR和Valois)的功能，并以这些因素作为分子切入点来测试两个量化N：C比率的模型。我建议通过探索Valois和dfmr1/CAPR突变表型的原因来研究调节果蝇早期胚胎N：C比值测量的机制。由于Valois和dfmr1已被证明与其他因素相互作用，我建议测试这些伙伴的功能丧失是否会发现更多的突变体，这些突变体在N：C比率测量中可能存在缺陷。有了这些突变体，我将使用Wieschaus实验室开发的一种区分N：C比率依赖和非独立影响的方法来测量这些缺陷对合子基因激活的时间控制的影响。最后，我将测试这类突变是否在物理和遗传上相互作用，以进一步了解这类表型突变背后的调控逻辑。我提出了两种通过翻译或染色质偶联机制来量化N：C比率的模型。通过进一步研究依赖于dfmr1/CAPR的母体mRNA表达的翻译控制和依赖于Valois的染色质修饰，我将检验两种测量N：C比率的候选机制。因此，该项目将扩大对早期发育调节系统和测量细胞大小的基本机制的理解。 与公共健康相关：细胞大小的测量是组织动态平衡的基本生物过程，起着防止细胞不受控制的生长的检查点的作用；但细胞监控其大小的机制鲜为人知。这项研究将在胚胎的背景下研究一种大小机制，核质比，当细胞大小测量调节早期发育中的主要转变时。这项工作旨在定义一种监测细胞大小和协调胚胎发育早期事件的基本机制。

DESCRIPTION (provided by applicant): One fundamental property of biological systems is the coordination of cellular proliferation with the regulation of cell size. This coordination ensures that cells grow to a minimum size prior to dividing and functions as a checkpoint to prevent unrestricted proliferation. While it is not fully understood how cells measure their size, it has lng been appreciated that the determination of cell size is coupled to the ratio of genomic DNA to cytoplasmic volume. This indicates that there exists a biological mechanism for measuring this nucleo-cytoplasmic (N:C) ratio, but the components of this mechanism are currently unknown. This research project proposes to use the study of the maternal to zygotic transition of the fruit fly Drosophila melanogaster as a model to understand further the mechanism of N:C ratio measurement. Early embryos couple the transition from maternal to zygotic control of development to the measurement of the N:C ratio. This transition results in global changes in embryonic gene expression, stemming, in part, from coordinated activities of both translation-regulatory and chromatin modifying mechanisms. However, a precise account of the mechanism underlying this coordination has been hampered by a paucity of mutants that specifically disrupt this process. The proposed investigation will examine the function of two recently identified Drosophila mutants with potential defects in N:C ratio measurement (dfmr1/capr and valois), and use these factors as a molecular entry point to test two models for quantifying the N:C ratio. I propose to investigate the mechanisms regulating the measurement of the N:C ratio in early Drosophila embryos by exploring the cause of the mutant phenotypes of valois and dfmr1/capr. Since valois and dfmr1 have been shown to interact with additional factors, I propose to test whether loss of function for these partners uncovers additional mutants with putative defects in N:C ratio measurement. With these mutants in hand, I will measure the effect of these defects on the temporal control of zygotic gene activation, using an assay developed in the Wieschaus lab that distinguishes N:C ratio-dependent and -independent effects. Finally, I will test whether mutants of this class interact physically and genetically in order to understand further the regulatory logic underlying this phenotypic class of mutants. I propose two models for quantification of the N:C ratio by translation- or chromatin-coupled mechanisms. By further investigation of dfmr1/capr-dependent translational control of maternal mRNA expression, and valois-dependent chromatin modification, I will examine two candidate mechanisms for measuring the N:C ratio. This project will therefore expand the understanding both of early developmental regulatory systems, and of a fundamental mechanism for measuring cellular size. PUBLIC HEALTH RELEVANCE: The measurement of cell size is a fundamental biological process that underlies tissue homeostasis, functioning as a checkpoint to prevent unregulated cell growth; but the mechanism whereby cells monitor their size is poorly understood. This research will study one sizing mechanism, the nucleo- cytoplasmic ratio, in the context of the embryo, when cell size measurement regulates a major transition during early development. This work aims to define a fundamental mechanism for monitoring cell size and coordinating the early events of embryonic development.