Final Report Summary - SIEAVD (Systems Imaging of Emerging Asymmetry in Vertebrate Development) After fertilization, the pre-implantation embryo will undergo several divisions before the first two different cell types can be identified in the 32-64 cell-blastocyst embryo: the trophectoderm (TE), giving rise to the placenta, and the inner cell mass (ICM) giving rise to the embryo. However, it remains strongly debated whether the acquisition of the first developmental differences preceding the induction of pluripotency in the ICM or extra-embryonic TE formation is a stochastic or a deterministic process.Given this controversy, reproducible molecular characterization of the emergence of asymmetry in the pre-implantation embryo resulting in the allocation of cells into the ICM or TE lineage is required. We are developing cutting-edge imaging technologies, assays, and reagents for sophisticated quantitative imaging analyses to systematically study development in vivo. By systematically applying advanced imaging tools, we anticipate identifying the earliest signs of developmental differences that can predict lineage-patterning events.Previously, we have established a fluorescence decay after photoactivation (FDAP) assay that allows for quantitative analysis of the kinetics of Oct4, a key transcription factor (TF) controlling pre-implantation development, fused to photoactivatable GFP (paGFP) at different stages of the developing mouse embryo. Strikingly, the quantitative analysis of Oct4–paGFP kinetics enabled us to predict pluripotency in the early mammalian embryo as early as at the 4-cell stage. We found that the population of cells with slow Oct4 kinetics and a high immobile fraction were much more likely to become a part of the eventual ICM of the developing embryo, while most cells that eventually make up the TE are derived from cells with fast Oct4 kinetics and a low immobile fraction.To further elucidate the elaborate protein and cell dynamics that underlie development, we expanded in vivo imaging to green-to-red photoconvertible fluorescent proteins (pcFPs). The key advantages of using pcFPs in developmental studies are: i) targeted proteins or cells can be visualized prior to photoconversion rendering them particularly amenable for high-throughput or high-content studies, and ii) both the non-photoconverted green protein as well as the photoconverted red protein populations can be tracked in parallel, which can provide more refined analysis than when using photoactivatable proteins.Until recently, spatially confined photoconversion using high-power, two-photon illumination was extremely inefficient. We now reported a unique optical mechanism, termed primed conversion, where dual-wavelength continuous-wave illumination results in pronounced photoconversion of fluorescent proteins. Notably, this two-step process requires significantly lower peak illumination intensities than nonlinear two-photon photomodulation, resulting in decreased phototoxicity when compared to i.e. nonlinear two-photon photoactivation of paGFP in our previous work.The segmentable signal from confined primed conversion opens up the possibility for non-invasive, high-contrast selection of targeted cells and/or proteins of interest, which will greatly facilitate systems imaging efforts during various developmental and disease processes. Here, we further developed confined primed conversion as a powerful and adequate tool to be able to decipher the emergence of asymmetry in early mammalian embryos.Specifically, we uncover the molecular mechanism of primed conversion. We employed this knowledge to create pr- (for primed convertible) variants of most known green-to-red pcFPs as well as several photoconvertible sensors and activity modulators. This achievement will dramatically expand the palette from previously only one protein (i.e. Dendra2) to all known anthozoan pcFPs and related sensors and effectors. Finally, we established primed convertible fast volumetric imaging as a superior imaging methodology to follow with high spatiotemporal precision all blastomeres across developmental time to unambiguously assign lineages and TF dynamics during early mouse embryo development.