Nuclear architecture and chromosomal dynamics in cleavage stage of development

描述(申请人提供)：在由母亲提供给卵子的基因产物控制的过程中，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个，小鼠的第一个，以及斑马鱼的前10个。斑马鱼母性效应突变体，树莓(BMB)，在早期细胞分裂阶段表现出专门的缺陷，类似于染色质Kid/Kinesin-10基因的母性效应小鼠突变体。这两个突变体都表现出一种独特的缺陷，即在发育的早期细胞分裂阶段，卵裂球表现出多个微核。由于Kid突变卵裂球的后期染色体紧凑性降低，后期和末期核膜的重组似乎独立地发生在Kid突变卵裂球的不同染色体团周围，导致多微核缺陷。微管相关蛋白CHICA与Kid结合，是Kid在细胞培养中定位于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的关系来扩展确保早期细胞分裂阶段单核形成的分子机制。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，可以用重要的荧光细胞周期标记物和延时显微镜在体内分析细胞分裂。这些研究对于理解人类生殖障碍和发展有效的生殖技术特别相关，因为通过体外受精产生的高质量的单精子胚胎中，约有15%在2-8-细胞期显示多核卵裂球。在目标1中，将确定BMB是否与Kid和CHICA在调节有丝分裂过程中的染色体动力学和防止多微核方面发挥类似的作用。这将通过体内延时显微镜检查BMB中的染色体动力学并将其与先前报道的KID和CHICA缺陷进行比较来完成。此外，还将通过基因敲除分析直接测试斑马鱼Kid的功能，以比较斑马鱼的Kid和BMB的功能。此外，斑马鱼CHICA功能的敲除将被执行，这可能为CHICA如何在发育中发挥作用提供一个体内模型。在目标2中，将通过电子显微镜超微结构分析来研究卵裂和卵裂后阶段的核结构及其动力学。在Aim3中，将确定BMB基因的分子性质。由于BMB不对应于KID或CHICA，它代表了一个新的因子，它专门在胚胎发育的早期母体调控的细胞分裂阶段发挥作用，因此将允许进一步阐明在这一独特的发育阶段调节染色体动力学和核结构的分子机制。 公共卫生相关性：拟议的研究对了解人类生殖障碍和开发有效的生殖技术特别相关，因为通过体外受精或胞浆内单精子注射产生的高质量单精子胚胎中，约有15%在2-8-细胞阶段显示多核卵裂球。对这些卵裂球的分析表明，染色体异常是经常发生的，因此，不建议将这些胚胎用于植入。了解模式生物早期胚胎细胞分裂阶段多微核的病因和分子基础，将为限制人类胚胎多核提供重要的参数和工具。

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.