Nuclear architecture and chromosomal dynamics in cleavage stage of development

发育裂解阶段的核结构和染色体动力学

• 批准号: 7773394
• 负责人: Mary C. Mullins
• 金额: $ 23.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7773394
• 关键词: Anaphase; Animal Model; Animals; Architecture; Biological Process; Cell Culture Techniques; Cell Cycle; Cell Nucleus; Cell division; Cell division phases; Cells; Chromosome abnormality; Chromosomes; Defect; Development; Disease; Electron Microscopy; Embryo; Embryonic Development; Ensure; Etiology; Exhibits; Fertilization in Vitro; Gene Expression; Genes; Genetic Transcription; Genome; Human; Injection of therapeutic agent; Kinesin; Mammals; Mediating; Microscopy; Microtubule-Associated Proteins; Mitosis; Mitotic spindle; Molecular; Mothers; Mus; Mutant Strains Mice; Nature; Nuclear; Nuclear Envelope; Phase; Phenotype; Play; Prevention; Process; Proteins; Regulation; Reliance; Reporting; Reproductive Technology; Role; Staging; Testing; Time; Zebrafish; base; blastocyst; blastomere structure; chromokinesin; egg; embryo stage 2; experimental analysis; implantation; in vivo; in vivo Model; loss of function; micronucleus; mutant; public health relevance; reproductive; sperm cell; telophase; tool; zygote

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.

描述（由申请人提供）：在由母亲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，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。斑马鱼母体效应突变体黑莓 (bmb) 在细胞分裂早期阶段表现出特定的缺陷，类似于小鼠染色因子 Kid/kinesin-10 基因的母体效应突变体。两种突变体都表现出独特的缺陷，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缩减少，后期后期和末期期间核膜的重新组装似乎独立发生在 Kid 突变体卵裂球中不同染色体团周围，从而导致多微核缺陷。微管相关蛋白 CHICA 与 Kid 相关，并且是 Kid 在细胞培养物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，扩展确保早期细胞分裂阶段单核化的分子机制。斑马鱼的早期卵裂阶段特别适合实验分析，因为胚胎在母体体外发育，是透明的且细胞很大，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。这里提出的研究与了解人类生殖疾病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 15% 在 2 至 8 细胞阶段显示出多核卵裂球。在目标 1 中，将确定 Bmb 是否以与 Kid 和 CHICA 类似的过程来调节有丝分裂期间的染色体动力学和预防多微核。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之前报道的 Kid 和 CHICA 缺陷进行比较来完成。此外，将通过敲低分析直接测试斑马鱼Kid的功能，以比较斑马鱼的Kid和bmb功能。此外，还将对斑马鱼 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体内模型。在目标 2 中，将通过电子显微镜超微结构分析在裂解和裂解后阶段研究核结构及其动力学。在 Aim3 中，将确定 bmb 基因的分子性质。由于 bmb 与 Kid 或 CHICA 不对应，它代表了一种新因子，专门在胚胎发育的早期、母体调节的细胞分裂阶段发挥作用，因此将有助于进一步阐明在此期间调节染色体动力学和核结构的分子机制。独特的发展阶段。 公共健康相关性：拟议的研究与了解人类生殖障碍和有效生殖技术的开发特别相关，因为通过体外受精或胞浆内单精子注射产生的优质单精子人类胚胎中，约 15% 在 2- 处显示多核卵裂球。至8细胞阶段。对此类卵裂球的分析表明，染色体异常经常发生，因此，不建议将这些胚胎用于植入。了解模型生物早期胚胎细胞分裂阶段多微核的病因学和分子基础将为限制人类胚胎中的多核提供重要的参数和工具。