Here, to investigate entry and exit form diapause I and II, we created a stable transgenic line in that expresses genetically encoded fluorescent reporters for cell cycle phases using the Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI) system that exploits the sequential degradation of fluorescent-tagged fragments of the cell cycle regulators Cdt1 and Geminin by the ubiquitin ligases SCF/Cdh1 and APC/C, respectively . corresponding author on a reasonable request. Abstract Background Annual killifishes are adapted to surviving Naphthoquine phosphate and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of diapause where their development is halted in anticipation of the months that have to elapse before their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a dispersed phase precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase Naphthoquine phosphate (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III). Developmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transition in living embryos. Results To visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell Naphthoquine phosphate cycle, respectively (Fluorescent Ubiquitination-based Cell Cycle Indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the G1 phase (diapause I). Thereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos. Conclusions Our study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state. Background Annual killifishes inhabit temporary habitats that are subject to periodic desiccations . In order to survive these extreme conditions, their eggs are laid in the soft substrate and remain encased in the dry mud where they are relatively protected from desiccation and can survive for prolonged periods during the dry season and regulate their development in anticipation of the ensuing rainy season. When their habitats are flooded, these embryos hatch, grow and mature rapidly and spawn the next generation before water evaporates [2C6]. This seasonal life cycle comprising embryonic arrest is widespread in arthropods from temperate climates, but it is unique among vertebrates. As an adaptation to seasonal water availability, embryonic development of annual killifishes deviates from canonical teleost development for three main distinctive traits. The first is a slow cell cycle during early cleavage. While embryos of non-annual teleost Naphthoquine phosphate fishes execute one cell division every 15C30?min during the first divisions after fertilization, the rate of early cell division in annual killifishes can reach almost 2?h . As a result, an annual killifish embryo can be still in Naphthoquine phosphate the blastula stage, while a non-annual killifish embryo fertilized at the same time has started somitogenesis. The second trait is the dispersion of epiblast cells during epiboly and a decoupling between epiboly and gastrulation. When epiboly starts, the epiblast cells delaminate, assume an amoeboid PIK3CB shape and migrate towards the other pole of the egg. This migration.