Nuclear architecture and chromosomal dynamics in cleavage stage of development

描述（由申请人提供）：在由母体提供给卵子的基因产物（所谓的母体基因产物）控制的过程中，有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），显示缺陷，特别是在这个早期的细胞分裂阶段，这类似于一个母体效应的小鼠突变体的chromokinesin Kid/kinesin-10基因。这两种突变体都显示出独特的缺陷，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突变体卵裂球中染色体的后期压实减少，后期和末期期间核膜的重新组装似乎独立地发生在Kid突变体卵裂球中不同的染色体质量周围，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，扩大在早期细胞分裂阶段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，允许用重要的荧光细胞周期标记物和延时显微镜对细胞卵裂进行体内分析。这里提出的研究是特别相关的了解人类生殖疾病和有效的生殖技术的发展，因为约15%的高质量的单精子体外受精产生的人类胚胎显示多核卵裂球在2- 8细胞阶段。在目标1中，将确定Bmb在有丝分裂期间染色体动力学调节和多微核预防中是否以与Kid和CHICA相似的过程发挥功能。这将通过在bmb中通过体内延时显微镜检查染色体动力学并将其与先前报道的Kid和CHICA缺陷进行比较来实现。此外，将通过敲低分析直接测试斑马鱼Kid的功能，以比较斑马鱼中的Kid和bmb功能。此外，将进行斑马鱼CHICA功能的敲低，可能为CHICA在发育中如何发挥作用提供体内模型。在目标2中，核结构及其动力学将通过电子显微镜超微结构分析在卵裂和卵裂后阶段进行研究。在Aim 3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，在胚胎发育的早期，母体调节的细胞分裂阶段特异性发挥作用，因此将允许进一步阐明在这个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：拟议的研究是特别相关的了解人类生殖疾病和有效的生殖技术的发展，因为约15%的高质量的单精子人类胚胎体外受精或胞质内单精子注射显示多核卵裂球在2至8细胞阶段。对这些卵裂球的分析表明，染色体异常是经常发生的，因此，这些胚胎不建议用于植入。了解模式生物早期胚胎细胞分裂阶段多微核形成的病因和分子基础，将为限制人类胚胎多核形成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.