Nuclear architecture and chromosomal dynamics in cleavage stage of development

描述（由申请人提供）：在母体提供给卵子的基因产物控制的过程中，即所谓的母体基因产物，是受精卵最早的细胞分裂。这些细胞分裂包括人类的前3次，小鼠的第一次和斑马鱼的前10次。斑马鱼的母体效应突变体brambleberry （bmb）在细胞分裂的早期阶段表现出特殊的缺陷，这类似于染色体驱动蛋白Kid/激酶10基因的母体效应小鼠突变体。这两种突变体都表现出一种独特的缺陷，即卵裂球在发育的早期细胞分裂阶段表现出多个微核。由于Kid突变体卵裂球后期染色体压实减少，后期和末期核膜的重组似乎在不同的染色体团块周围独立发生，导致多微核缺陷。微管相关蛋白CHICA与Kid相关，是Kid在细胞培养中定位到有丝分裂纺锤体所必需的。在这里，我们将通过分析bmb突变体及其与Kid和CHICA的关系，进一步阐述在细胞分裂早期确保单核化的分子机制。斑马鱼的早期卵裂阶段特别适合实验分析，因为胚胎在母体外发育，是透明的，细胞很大，允许使用重要的荧光细胞周期标记和延时显微镜对细胞卵裂进行体内分析。这里提出的研究与理解人类生殖障碍和有效生殖技术的发展特别相关，因为通过体外受精产生的优质单精子人类胚胎中约有15%在2至8细胞阶段显示多核卵裂球。在Aim 1中，我们将确定Bmb在有丝分裂过程中对染色体动力学的调节和多微核的预防过程是否与Kid和CHICA类似。这将通过bmb体内延时显微镜检查染色体动力学来完成，并将其与先前报道的Kid和CHICA缺陷进行比较。此外，我们将通过敲低分析直接检测斑马鱼Kid的功能，比较斑马鱼Kid和bmb的功能。此外，还将对斑马鱼的CHICA功能进行敲除，这可能为CHICA在发育过程中如何发挥作用提供一个体内模型。在目标2中，核结构及其动力学将通过电子显微镜和超微结构分析在裂解和后裂解阶段进行研究。在Aim3中，将确定bmb基因的分子性质。由于bmb不对应于Kid或CHICA，它代表了一种新的因子，专门在胚胎发育的早期，母亲调节的细胞分裂阶段起作用，因此将允许进一步阐明在这一独特发育阶段调节染色体动力学和核结构的分子机制。

DESCRIPTION (provided by applicant): Among the processes controlled by gene products supplied to the egg by the mother, so called maternal gene products, are the earliest cell divisions of the zygote. These cell divisions encompass the first 3 in humans, the first one in mouse, and the first 10 in zebrafish. A zebrafish maternal-effect mutant, brambleberry (bmb), displays defects specifically during this early cell division stage, which resemble a maternal-effect mouse mutant of the chromokinesin Kid/kinesin-10 gene. Both mutants display a unique defect whereby blastomeres exhibit multiple, micronuclei during this early cell division phase of development. Due to reduced anaphase compaction of chromosomes in Kid mutant blastomeres, reassembly of the nuclear envelope during late anaphase and telophase appears to occur independently around distinct chromosomal masses in Kid mutant blastomeres, causing the multi-micronuclei defect. The microtubule associated protein CHICA associates with Kid and is required for Kid's localization to the mitotic spindle in cell culture. Here, we will expand upon the molecular mechanisms that ensure mononucleation during early cell division stages through analysis of the bmb mutant and its relationship to Kid and CHICA. The early cleavage stage in zebrafish is particularly amenable to experimental analysis, as the embryo develops outside the mother, is transparent and the cells are large, allowing in vivo analysis of cell cleavage with vital fluorescent cell cycle markers and time-lapse microscopy. The studies proposed here are particularly relevant to understanding human reproductive disorders and the development of effective reproductive technology, since about 15% of good quality monospermic human embryos produced by in vitro fertilization display multinucleated blastomeres at the 2- to 8-cell stage. In Aim 1, it will be determined if Bmb functions in a similar process as Kid and CHICA in the regulation of chromosomal dynamics during mitosis and the prevention of multi-micronucleation. This will be accomplished by examining chromosomal dynamics via in vivo time-lapse microscopy in bmb and comparing it to the previously reported Kid and CHICA defects. Furthermore, the function of zebrafish Kid will be directly tested by knockdown analysis to compare Kid and bmb functions in zebrafish. In addition, knockdown of zebrafish CHICA function will be performed, potentially providing an in vivo model for how CHICA functions in development. In Aim 2, nuclear architecture and its dynamics will be investigated during cleavage and post cleavage stages through electron microscopy ultrastructural analysis. In Aim3, the molecular nature of the bmb gene will be determined. Since bmb does not correspond to Kid or CHICA, it represents a new factor functioning specifically during the early, maternally-regulated cell division phase of embryonic development, and thus will allow the further elucidation of the molecular mechanisms regulating chromosomal dynamics and nuclear architecture during this unique phase of development. PUBLIC HEALTH RELEVANCE: The proposed studies are particularly relevant to understanding human reproductive disorders and the development of effective reproductive technology, since about 15% of good quality monospermic human embryos produced by in vitro fertilization or intra cytoplasmic sperm injection display multinucleated blastomeres at the 2- to 8-cell stage. Analysis of such blastomeres has shown that chromosomal abnormalities are a frequent occurrence and as such, these embryos are not advised for use in implantation. Understanding the etiology and molecular basis of multi-micronucleation during early embryonic cell division stages in model organisms will provide important parameters and tools to limit multinucleation in human embryos.