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.

Project Narrative Disruptions to the oocyte-to-embryo transition contribute to infertility and miscarriage in humans. To explore regulation of these events, we developed a reporter to visualize the universally conserved calcium wave that occurs at the moment of fertilization in a living organism. Now, we propose to use this tool to investigate the underlying signaling pathways that coordinate this critical period of development.