FuChi: A cell cycle biosensor for investigating cell-cycle kinetics during avian development

The ability to monitor the proliferative status in live cells both in vitro and in vivo over time has revolutionised our understanding of development, growth and disease. This was first made possible by fluorescent ubiquitination-based cell cycle indicator (Fucci) technology, which distinguishes specific cell cycle phases through the reciprocal degradation of fluorescently tagged, truncated forms of human CDT1 and GMNN proteins. Fucci genetic systems have been successfully implemented in transgenic mice, zebrafish, and axolotls. To date, no viable, stably expressing Fucci line has been developed in an avian species. Although a range of continuously improving Fucci constructs have been developed in recent years, existing in vivo Fucci models remain limited because they rely on older reporter technology that fails to distinguish cells in S, G2, and M phases or to label cells in early G1. As a result, these models can be challenging to interpret and their utility for continuous cell tracking and precise analysis of cell-cycle dynamics is limited. Here, we introduce FuChi, a multicistronic Fucci-expressing chicken line incorporating a newly optimised reporter construct comprising of an mCerulean-tagged Histone H1.0 linker protein fused via a self-cleaving 2A peptide to the tandem Fucci(CA) cell cycle biosensor, with additional epitope tags included for detection in fixed tissues. We show that this system accurately discriminates and tracks cells in G1, S, G2, and M phases both in vitro and in vivo , enabling faithful visualisation of cell cycle status in intact tissues and organs. Using FuChi embryos, we mapped proliferation dynamics across developing tissues, analysed cell cycle states of migrating cells, and performed live imaging of early embryos. These latter experiments revealed that transition from S phase may be a key morphogenetic event during gastrulation as mesendoderm cells egress from the primitive streak to form embryonic structures including the prechordal plate. Pairing this advanced reporter with the intrinsic experimental advantages of the chicken embryo, positions FuChi as a premier in vivo system for studying cell-cycle kinetics in development, delivering clear technological improvements over current Fucci models. FuChi chickens provide a powerful new resource for studying embryonic development, organ growth, tissue homeostasis, disease processes, and infection responses.