The Role of Calcium Transients in Fertilization and Early Embryogenesis in C. elegans

钙瞬变在秀丽隐杆线虫受精和早期胚胎发生中的作用

• 批准号: 10654250
• 负责人: Karen S Kim Guisbert
• 金额: $ 43.04万
• 依托单位: FLORIDA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654250
• 关键词: Academic Research Enhancement Awards; Address; Animals; Biological Models; Caenorhabditis elegans; Calcium; Calcium Oscillations; Calcium Signaling; Calcium Spikes; Cell Communication; Cell division; Cells; Communication; Competence; Complement; Complex; Consumption; Cytology; Data; Defect; Dependence; Development; Developmental Biology; Dyes; Education; Educational process of instructing; Embryo; Embryo Research; Embryonic Development; Event; Fertility; Fertilization; Florida; Genetic; Germ Cells; Goals; Gonadal structure; Human; Human Development; Image; In Vitro; Infertility; Injections; Knowledge; Learning; Marriage; Meiosis; Microscopy; Mitotic; Molecular; Nature; Neurons; Oocytes; Optics; Organism; Ovulation; Pattern; Phase; Phenotype; Physiology; Process; RNA interference screen; Regulation; Reporter; Reporting; Reproductive Medicine; Research; Research Infrastructure; Research Project Grants; Role; Series; Signal Pathway; Signal Transduction; Slice; Specificity; Spontaneous abortion; Students; System; Techniques; Technology; Testing; Time; Visualization; biochemical tools; calcium indicator; cell type; course redesign; critical period; design; egg; experimental study; gastrulation; improved; in vivo; innovation; insight; knock-down; laboratory experience; model organism; novel; prevent; response; sperm cell; student participation; tool; undergraduate research experience; undergraduate student

Project Summary Human development begins with an internal fertilization event. However, internal fertilization is notoriously difficult to visualize in vivo. Taking advantage of the universally conserved calcium response that is a hallmark of sperm-egg fusion in all species, we have adapted the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization inside a living animal, C. elegans. Our system produces a robust signal that recapitulates the previously reported, biphasic nature of the calcium wave and has no deleterious effects on worm physiology or fecundity. This reporter is a powerful new tool to facilitate our long-term goal to define cell-cell communication and coordination during the events of the oocyte-to-embryo transition and early embryogenesis. The rationale is that visualization of the calcium transients, in conjunction with genetic and molecular tools, will allow an unprecedented opportunity to dissect early embryogenesis inside a living animal. Completion of Specific Aim 1 will define novel intercellular regulation and coordination that occurs during the oocyte-to- embryo transition by examining our reporter in a background that forces both a fertilization-competent and a fertilization-incompetent oocyte to be ovulated at the same time. Specific Aim 2 will establish the cell-type specificity and timing of calcium transients during early embryogenesis that have never before been observed in C. elegans. Specific Aim 3 will incorporate a targeted RNAi screen for OET regulators into a redesigned course-based undergraduate research experience (CURE) lab. This will synergistically propel OET research and improve undergraduate Developmental Biology education at Florida Tech. The central innovation of this proposal is the marriage of superior calcium imaging during early embryogenesis combined with a model system that has superlative genetics and a well-ordered gonad. This proposed research is significant because it will enable new insights into the timing and regulation of the OET and early embryogenesis in an intact living animal and could led to advances in reproductive medicine. Importantly, this proposal will satisfy key objectives of the R15 mechanism and the Notice of Special Intent (NOT-HD-19-036) by 1) significantly enhancing exposure of students to developmental biology research and 2) strengthening the research infrastructure at the Florida Institute of Technology.

项目摘要 人类发展始于内部施肥事件。但是，内部 众所周知，很难在体内形象化受精。利用普遍的优势 保守的钙反应是所有物种中精子融合的标志，我们 已经适应了遗传编码的钙指示器JGCAMP7s荧光 指示活动物C.秀拉斯杆菌中受精的时刻。我们的系统 产生强大的信号，该信号概括了先前报道的，双相的性质 钙波，对蠕虫生理或繁殖力没有有害影响。这 记者是一个有力的新工具，可以促进我们定义细胞细胞的长期目标 卵母细胞到embryo过渡的事件中的沟通和协调 早期胚胎发生。理由是钙瞬变的可视化，在 与遗传和分子工具的结合，将使前所未有的机会 解剖活动物中的早期胚胎发生。特定目标的完成1将 定义在卵母细胞到卵母细胞期间发生的新型细胞间调节和协调 胚胎过渡通过检查我们的记者在迫使两者的背景下进行过渡 施肥能力和施肥不足的卵母细胞在同一 时间。特定的目标2将建立细胞类型的特异性和钙瞬变的时机 在早期的胚胎发生期间，在秀丽隐杆线虫中从未观察过。 特定的目标3将将OET调节器的靶向RNAi屏幕纳入一个 重新设计的基于课程的本科研究经验（CURE）实验室。这会 协同推动OET研究并改善本科发展生物学 佛罗里达理工大学的教育。该提议的中心创新是 早期胚胎发生过程中的上钙成像与模型系统结合 具有最高级的遗传学和井井有条的性腺。这项拟议的研究很重要 因为它将能够对OET和早期的时机和调节进行新的见解 完整的活动物中的胚胎发生，并可能导致生殖进展 药品。重要的是，该建议将满足R15机制的关键目标，并 特殊意图的通知（非HD-19-036）通过1）显着增强 学生进行发育生物学研究和2）加强研究 佛罗里达理工学院的基础设施。