Nuclear architecture and chromosomal dynamics in cleavage stage of development

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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.