MITO-Luc/GFP zebrafish model to assess spatial and temporal evolution of cell proliferation in vivo

Luisa De Latouliere,Ombretta Pozzoli,Aymone Gurtner,Federica Pisati,Isabella Manni,Giulia Piaggio,Lucrezia Pacello,Laura Ferrari,Gianluca Deflorian,Maria Grazia Totaro,Maurizio C Capogrossi,Luigi Aurisicchio,Emanuele Marra

doi:10.1038/s41598-020-79530-5

Abstract

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo.

Highlights

We developed a novel reporter transgenic zebrafish model called MITO-Luc/green fluorescent protein (GFP) zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation nuclear transcription factor Y (NF-Y)
In vitro luciferase activity assay performed in lysates showed 80-fold increased transcriptional activity in pT2KXIGΔin-cyclin B2-Luc/GFP compared to pT2KXIGΔin-Luc/GFP injected embryos (Fig. 1B)
We investigated whether inhibition of cell proliferation was associated with inhibition of luciferase expression in MITO-Luc/GFP1 zebrafish model

Summary

Introduction

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. Non-invasive BLI has been widely used in mice to monitor gene expression[18], bacterial and viral infections[19], as well as cell proliferation and transformation[20] This technique allows the serial quantification of spatial and temporal evolution of the process of interest in the same animal[19]. We previously generated a mouse model, called MITO-Luc reporter mouse, to visualize proliferation events in live animals In these lines the luciferase reporter gene is under the control of a cyclin B2 minimal promoter containing three CCAAT boxes conserved between mouse and human and tightly regulated by the nuclear transcription factor Y (NF-Y)[22]. NF-Y is conserved in evolution and its activity is required for transcription of regulatory genes responsible for cell cycle progression, supporting cell proliferation[24–33]

Methods

Results

Conclusion