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Angiogenesis

, Volume 21, Issue 2, pp 349–361 | Cite as

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system

  • Katia Herz
  • Alexandra Becker
  • Chenyue Shi
  • Masatsugo Ema
  • Satoru Takahashi
  • Michael Potente
  • Michael Hesse
  • Bernd K. Fleischmann
  • Daniela Wenzel
Original Paper

Abstract

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.

Keywords

Endothelial cell Proliferation Angiogenesis Anillin Cell cycle 

Notes

Acknowledgements

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.

Author contributions

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.

Funding

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.

Supplementary material

10456_2018_9601_MOESM1_ESM.pdf (13.7 mb)
Supplementary material 1 (PDF 14054 kb)
10456_2018_9601_MOESM2_ESM.avi (30 mb)
Supplementary material 2 (AVI 30766 kb)

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Physiology I, Life and Brain Center, Medical FacultyUniversity of BonnBonnGermany
  2. 2.Angiogenesis and Metabolism LaboratoryMax Planck Institute for Heart and Lung ResearchBad NauheimGermany
  3. 3.Department of Stem Cells and Human Disease Models, Research Center for Animal Life ScienceShiga University of Medical ScienceOtsuJapan
  4. 4.Department of Anatomy and Embryology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human SciencesUniversity of TsukubaTsukubaJapan
  5. 5.International Institute of Molecular and Cell BiologyWarsawPoland
  6. 6.DZHK (German Center for Cardiovascular Research)BerlinGermany

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