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

项目摘要 人类的发展始于一个内部受精事件。然而，内部 众所周知，受精在体内难以观察。利用普遍存在的 保守的钙反应是所有物种精卵融合的标志，我们 已经将遗传编码的钙指示剂jGCaMP 7s调整为荧光 表明在活体动物体内受精的时刻，C。优雅我们的系统 产生了一个强大的信号，概括了以前报道的，双相的性质， 钙波，对蠕虫的生理和繁殖力无不良影响。这 报告基因是一个强大的新工具，有助于我们实现定义细胞-细胞的长期目标 在卵母细胞向胚胎过渡期间的沟通和协调， 早期胚胎发生基本原理是钙瞬变的可视化， 与遗传和分子工具相结合，将提供前所未有的机会， 解剖活体动物体内的早期胚胎发育。完成具体目标1将 定义了卵母细胞到卵母细胞过程中发生的新的细胞间调节和协调， 胚胎过渡通过研究我们的记者在一个背景下， 受精能力和不能受精的卵母细胞同时排卵 时间具体目标2将确定细胞类型特异性和钙瞬变的时间 在早期胚胎发生过程中，这在C.优雅 Specific Aim 3将把OET调节剂的靶向RNAi筛选纳入到一个 重新设计的基于课程的本科生研究经验（CURE）实验室。这将 协同推进OET研究提高本科生发育生物学水平 在佛罗里达理工大学接受教育这一建议的核心创新是 在早期胚胎发生过程中的上级钙成像与模型系统相结合， 拥有最好的基因和有序的性腺这项研究具有重要意义 因为它将使新的见解的时间和监管的OET和早期 胚胎发生在一个完整的活的动物，并可能导致生殖的进步， 药重要的是，该提案将满足R15机制的关键目标， 特别意图通知（NOT-HD-19-036）通过1）显著提高 2）加强研究 佛罗里达理工学院的基础设施。

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.