Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system
Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP+ signals specifically in Ki67+/PECAM+ endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.
KeywordsEndothelial cell Proliferation Angiogenesis Anillin Cell cycle
We thank A. Nagy (Toronto, Canada) for providing G4 mouse ES cells. Moreover, we would like to acknowledge P. Freitag (University of Bonn, Germany) for excellent technical assistance and D. Korzus (University of Bonn) for help with determination of estrus cycle.
KH has generated Flt-1/eGFP-anillin mice, performed expression analysis of eGFP-anillin at different stages and performed live monitoring including quantitative analyses, AR has acquired pictures of sections from embryonic and adult tissue and established live monitoring of the embryos, CS has acquired data from hindbrains and retinas, ST and ME have generated Flt-1/tdsred mice and contributed to the writing of the manuscript, MP has supervised hindbrain and retina analysis and contributed to the writing of the manuscript, MH has generated Flt-1/eGFP-anillin mice by complementation of ES cells with diploid mouse embryos, BF has contributed to the design of the study and the writing of the manuscript, DW has designed the study, supervised analysis and wrote the manuscript.
M.P. is supported by the Max Planck Society, the European Research Council (ERC) Starting Grant ANGIOMET (311546), the Deutsche Forschungsgemeinschaft (SFB 834), the Excellence Cluster Cardiopulmonary System (EXC 147/1), the LOEWE Grant Ub-Net, the DZHK (German Center for Cardiovascular Research), the Stiftung Charité, and the European Molecular Biology Organization Young Investigator Programme.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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