Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Pharmacological manipulation of blood and lymphatic vascularization in ex vivo–cultured mouse embryos

Nature Protocols volume 7, pages 1970–1982 (2012)Cite this article

Subjects

Abstract

Formation of new blood and lymphatic vessels is involved in many physiological and pathological processes, including organ and tumor growth, cancer cell metastasis, fluid drainage and lymphedema. Therefore, the ability to manipulate vascularization in a mammalian system is of particular interest to researchers. Here we describe a method for pharmacological manipulation of de novo and sprouting blood and lymphatic vascular development in ex vivo–cultured mouse embryos. The described protocol can also be used to evaluate the properties of pharmacological agents in growing mammalian tissues and to manipulate other developmental processes. The whole procedure, from embryo isolation to image quantification, takes 3–5 d, depending on the analysis and age of the embryos.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Flowchart of ex vivo manipulation and cultivation of mouse embryos.
Figure 2: Equipment setup and injection sites in mice at different embryonic stages.
Figure 3: Typical confocal images of blood and lymphatic vascular development after microinjection in whole-embryo culture (WEC).

Similar content being viewed by others

References

  1. Ferrara, N. et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439–442 (1996).

    Article  CAS  Google Scholar 

  2. Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439 (1996).

    Article  CAS  Google Scholar 

  3. Duarte, A. et al. Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev. 18, 2474–2478 (2004).

    Article  CAS  Google Scholar 

  4. Leveen, P. et al. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 8, 1875–1887 (1994).

    Article  CAS  Google Scholar 

  5. Lindahl, P., Johansson, B.R., Leveen, P. & Betsholtz, C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242–245 (1997).

    Article  CAS  Google Scholar 

  6. Strilic, B. et al. The molecular basis of vascular lumen formation in the developing mouse aorta. Dev. Cell 17, 505–515 (2009).

    Article  CAS  Google Scholar 

  7. Strilic, B. et al. Electrostatic cell-surface repulsion initiates lumen formation in developing blood vessels. Curr. Biol. 20, 2003–2009 (2010).

    Article  CAS  Google Scholar 

  8. Randhawa, P.K. et al. The Ras activator RasGRP3 mediates diabetes-induced embryonic defects and affects endothelial cell migration. Circ. Res. 108, 1199–1208 (2011).

    Article  CAS  Google Scholar 

  9. Nagase, M., Nagase, T., Koshima, I. & Fujita, T. Critical time window of hedgehog-dependent angiogenesis in murine yolk sac. Microvasc. Res. 71, 85–90 (2006).

    Article  CAS  Google Scholar 

  10. Downs, K.M., Hellman, E.R., McHugh, J., Barrickman, K. & Inman, K.E. Investigation into a role for the primitive streak in development of the murine allantois. Development 131, 37–55 (2004).

    Article  CAS  Google Scholar 

  11. Kawasaki, K. et al. Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells. J. Cell Biol. 181, 131–141 (2008).

    Article  CAS  Google Scholar 

  12. Bohnsack, B.L., Lai, L., Dolle, P. & Hirschi, K.K. Signaling hierarchy downstream of retinoic acid that independently regulates vascular remodeling and endothelial cell proliferation. Genes Dev. 18, 1345–1358 (2004).

    Article  CAS  Google Scholar 

  13. Planas-Paz, L. et al. Mechanoinduction of lymph vessel expansion. EMBO J. 31, 788–804 (2011).

    Article  Google Scholar 

  14. Xu, K. et al. Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling. Dev. Cell 20, 526–539 (2011).

    Article  CAS  Google Scholar 

  15. Parker, L.H. et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 428, 754–758 (2004).

    Article  CAS  Google Scholar 

  16. Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 (2003).

    Article  CAS  Google Scholar 

  17. Wigle, J.T. & Oliver, G. Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769–778 (1999).

    Article  CAS  Google Scholar 

  18. Karkkainen, M.J. et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5, 74–80 (2004).

    Article  CAS  Google Scholar 

  19. Kiefer, F. & Adams, R.H. Lymphatic endothelial differentiation: start out with Sox—carry on with Prox. Genome Biol. 9, 243 (2008).

    Article  Google Scholar 

  20. Makinen, T., Norrmen, C. & Petrova, T.V. Molecular mechanisms of lymphatic vascular development. Cell Mol. Life Sci. 64, 1915–1929 (2007).

    Article  CAS  Google Scholar 

  21. Böhmer, R. et al. Regulation of developmental lymphangiogenesis by Syk+ leukocytes. Dev. Cell 18, 437–449 (2010).

    Article  Google Scholar 

  22. Tammela, T. & Alitalo, K. Lymphangiogenesis: molecular mechanisms and future promise. Cell 140, 460–476 (2010).

    Article  CAS  Google Scholar 

  23. Kälin, R.E., Banziger-Tobler, N.E., Detmar, M. & Brändli, A.W. An in vivo chemical library screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis and lymphangiogenesis. Blood 114, 1110–1122 (2009).

    Article  Google Scholar 

  24. Hellstrom, M., Kalen, M., Lindahl, P., Abramsson, A. & Betsholtz, C. Role of PDGF-B and PDGFR-β in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126, 3047–3055 (1999).

    CAS  PubMed  Google Scholar 

  25. Norrmen, C. et al. FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1. J. Cell Biol. 185, 439–457 (2009).

    Article  CAS  Google Scholar 

  26. Sabine, A. et al. Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation. Dev. Cell 22, 430–445 (2012).

    Article  CAS  Google Scholar 

  27. Sasaki, T. et al. Regulation of hematopoietic cell clusters in the placental niche through SCF/Kit signaling in embryonic mouse. Development 137, 3941–3952 (2010).

    Article  CAS  Google Scholar 

  28. Hang, C.T. et al. Chromatin regulation by Brg1 underlies heart muscle development and disease. Nature 466, 62–67 (2010).

    Article  CAS  Google Scholar 

  29. Chang, C.P. et al. A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis. Cell 118, 649–663 (2004).

    Article  CAS  Google Scholar 

  30. Miki, R. et al. Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos. Mech. Dev. 128, 597–609 (2012).

    Article  CAS  Google Scholar 

  31. Tremblay, K.D. & Zaret, K.S. Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues. Dev. Biol. 280, 87–99 (2005).

    Article  CAS  Google Scholar 

  32. Noda, T. et al. Restriction of Wnt signaling in the dorsal otocyst determines semicircular canal formation in the mouse embryo. Dev. Biol. 362, 83–93 (2012).

    Article  CAS  Google Scholar 

  33. Calegari, F. & Huttner, W.B. An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis. J. Cell Sci. 116, 4947–4955 (2003).

    Article  CAS  Google Scholar 

  34. Inoue, T. & Krumlauf, R. An impulse to the brain—using in vivo electroporation. Nat. Neurosci. 4 (suppl.): 1156–1158 (2001).

    Article  CAS  Google Scholar 

  35. Gray, J. & Ross, M.E. Neural tube closure in mouse whole embryo culture. J. Vis. Exp. published online, doi:10.3791/3132 (2011).

  36. Takahashi, M., Nomura, T. & Osumi, N. Transferring genes into cultured mammalian embryos by electroporation. Dev. Growth Differ. 50, 485–497 (2008).

    Article  CAS  Google Scholar 

  37. Winn, L.M. & Tung, E.W. Assessment of embryotoxicity using mouse embryo culture. Methods Mol. Biol. 550, 241–249 (2009).

    Article  CAS  Google Scholar 

  38. Jones, E.A. et al. Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture. Genesis 34, 228–235 (2002).

    Article  CAS  Google Scholar 

  39. Kwon, G.S. et al. Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development. Dev. Dyn. 235, 2549–2558 (2006).

    Article  CAS  Google Scholar 

  40. Schmidt, M. et al. EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution. Development 134, 2913–2923 (2007).

    Article  CAS  Google Scholar 

  41. Boisset, J.C., Andrieu-Soler, C., van Cappellen, W.A., Clapes, T. & Robin, C. Ex vivo time-lapse confocal imaging of the mouse embryo aorta. Nat. Protoc. 6, 1792–1805 (2011).

    Article  CAS  Google Scholar 

  42. Drake, C.J. & Fleming, P.A. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95, 1671–1679 (2000).

    CAS  PubMed  Google Scholar 

  43. Veikkola, T. et al. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. 20, 1223–1231 (2001).

    Article  CAS  Google Scholar 

  44. Skobe, M. et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat. Med. 7, 192–198 (2001).

    Article  CAS  Google Scholar 

  45. Garcia, M.D., Udan, R.S., Hadjantonakis, A.K. & Dickinson, M.E. Preparation of rat serum for culturing mouse embryos. Cold Spring Harb. Protoc. 2011, 391–393 (2011).

    Google Scholar 

  46. Schulte-Merker, S., Sabine, A. & Petrova, T.V. Lymphatic vascular morphogenesis in development, physiology, and disease. J. Cell Biol. 193, 607–618 (2011).

    Article  CAS  Google Scholar 

  47. Carmeliet, P. et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat. Med. 7, 575–583 (2001).

    Article  CAS  Google Scholar 

  48. Akerman, S. et al. Microflow of fluorescently labelled red blood cells in tumours expressing single isoforms of VEGF and their response to vascular targeting agents. Med. Eng. Phys. 33, 805–809 (2011).

    Article  Google Scholar 

  49. Fong, T.A. et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 59, 99–106 (1999).

    CAS  PubMed  Google Scholar 

  50. Karpanen, T. et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res. 61, 1786–1790 (2001).

    CAS  PubMed  Google Scholar 

  51. Bakkers, J. Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc. Res. 91, 279–288 (2011).

    Article  CAS  Google Scholar 

  52. Jin, S.W., Beis, D., Mitchell, T., Chen, J.N. & Stainier, D.Y. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199–5209 (2005).

    Article  CAS  Google Scholar 

  53. Patan, S. Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J. Neurooncol. 50, 1–15 (2000).

    Article  CAS  Google Scholar 

  54. Thisse, C. & Zon, L.I. Organogenesis—heart and blood formation from the zebrafish point of view. Science 295, 457–462 (2002).

    Article  CAS  Google Scholar 

  55. Mendel, D.B. et al. The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function. Clin. Cancer Res. 6, 4848–4858 (2000).

    CAS  PubMed  Google Scholar 

  56. Finnerty, H. et al. Molecular cloning of murine FLT and FLT4. Oncogene 8, 2293–2298 (1993).

    CAS  PubMed  Google Scholar 

  57. Abramoff, M.D., Magalhaes, P.J. & Ram, S.J. Image processing with ImageJ. Biophotonics International 11, 36–42 (2004).

    Google Scholar 

  58. Walls, J.R., Coultas, L., Rossant, J. & Henkelman, R.M. Three-dimensional analysis of vascular development in the mouse embryo. PLoS ONE 3, e2853 (2008).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to M. Gearing for proofreading the manuscript and to the other members of the Lammert laboratory for valuable discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) LA 1216/4-1 and 5-1, and by the Collaborative Research Centre (CRC) 974.

Author information

Author notes

  1. Martin Zeeb and Jennifer Axnick: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Metabolic Physiology, Heinrich-Heine University Duesseldorf, Duesseldorf, Germany

    Martin Zeeb, Jennifer Axnick, Lara Planas-Paz, Thorsten Hartmann & Eckhard Lammert

  2. Department of Pharmacology, Max Planck Institute for Heart and Lung Research, W.G. Kerckhoff-Institute, Bad Nauheim, Germany

    Boris Strilic

  3. German Diabetes Center (DDZ), Paul Langerhans Group for Beta Cell Biology, Duesseldorf, Germany

    Eckhard Lammert

Authors
  1. Martin Zeeb
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer Axnick
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lara Planas-Paz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thorsten Hartmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Boris Strilic
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eckhard Lammert
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.Z., J.A. and E.L. wrote the text. M.Z. and J.A. prepared the figures. M.Z., J.A., B.S. and T.H. performed the blood vessel experiments. L.P.-P. and J.A. performed the lymphatic vessel experiments.

Corresponding author

Correspondence to Eckhard Lammert.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Images to illustrate important steps of skin isolation as described in step 20C. (PDF 212 kb)

Supplementary Figure 2

Transversal sections through mouse embryos after microinjection and whole embryo culture (WEC) illustrating the local effects of injected substances. (PDF 1063 kb)

Supplementary Table 1

Survival rates of long-term WEC in M16. (PDF 38 kb)

Supplementary Table 2

Range of heart rates after WEC. (PDF 57 kb)

Supplementary Video 1

Uterus separation, isolation and injection of E8.0 mouse embryos are shown together with common mistakes. (MOV 31388 kb)

Supplementary Video 2

Uterus separation, isolation and injection of E8.75 mouse embryos are shown together with common mistakes. (MOV 14856 kb)

Supplementary Video 3

Uterus separation, isolation and injection of E11.5 mouse embryos are shown together with common mistakes. (MOV 18778 kb)

Supplementary Video 4

Uterus separation, isolation and injection of E15.5 mouse embryos are shown together with common mistakes. (MOV 14729 kb)

Supplementary Video 5

Heartbeat movie of an E8.5 mouse embryo after 12 h of WEC. (MOV 6926 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zeeb, M., Axnick, J., Planas-Paz, L. et al. Pharmacological manipulation of blood and lymphatic vascularization in ex vivo–cultured mouse embryos. Nat Protoc 7, 1970–1982 (2012). https://doi.org/10.1038/nprot.2012.120

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.120

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research