Long-term labelling and tracing of endodermal cells using a perpetual cycling Gal4-UAS system

Cell labelling and lineage tracing are indispensable tools in developmental biology, offering powerful means with which to visualise and understand the complex dynamics of cell populations during embryogenesis. Traditional cell labelling relies heavily on signal stability, promoter strength and stage specificity, limiting its application in long-term tracing. In this report, we optimise and reconfigure a perpetual cycling Gal4-UAS system employing a previously unreported Gal4 fusion protein and the autoregulatory Gal4 expression loop. As validated through heat-shock induction, this configuration ensures sustained transcription of reporter genes in target cells and their descendant cells while minimising cytotoxicity, thereby achieving long-term labelling and tracing. Further exploiting this system, we generate zebrafish transgenic lines with continuous fluorescent labelling specific to the endoderm, and demonstrate its effectiveness in long-term tracing by showing the progression of endoderm development from embryo to adult, providing visualisation of endodermal cells and their derived tissues. This continuous labelling and tracing strategy can span the entire process of endodermal differentiation, from progenitor cells to mature functional cells, and is applicable to studying endoderm patterning and organogenesis.