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Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

Nature Protocols volume 7, pages 421–431 (2012)Cite this article

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Abstract

We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5–11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.

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Figure 1
Figure 2: Increasing tissue transparency using BABB solution.
Figure 3: Embryo trimming.
Figure 4: Slide preparation.
Figure 5: Three-dimensional reconstruction of the dorsal aorta region stained with c-Kit (green)- and CD31 (magenta)-specific antibodies at E10.5 (35 somite pairs).
Figure 6: Quantification procedure of c-Kit+ hematopoietic cluster cells in the dorsal aorta.
Figure 7: Three-dimensional reconstructed pictures at low and high magnifications.
Figure 8: Three-dimensional reconstructed pictures showing nuclear expression of phosphohistone H3.

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References

  1. Dzierzak, E. & Speck, N.A. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat. Immunol. 9, 129–136 (2008).

    Article  CAS  Google Scholar 

  2. Medvinsky, A., Rybtsov, S. & Taoudi, S. Embryonic origin of the adult hematopoietic system: advances and questions. Development 138, 1017–1031 (2011).

    Article  CAS  Google Scholar 

  3. Marshall, C.J. & Thrasher, A.J. The embryonic origins of human haematopoiesis. Br. J. Haematol. 112, 838–850 (2001).

    Article  CAS  Google Scholar 

  4. Tavian, M. et al. Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 87, 67–72 (1996).

    CAS  PubMed  Google Scholar 

  5. Gard, D.L. Confocal immunofluorescence microscopy of microtubules in amphibian oocytes and eggs. Methods Cell Biol. 38, 241–264 (1993).

    Article  CAS  Google Scholar 

  6. Zucker, R.M., Hunter, E.S. & Rogers, J.M. (eds.) Confocal Laser Scanning Microscopy of Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos 191–202 (Humana Press, 1999).

  7. Yokomizo, T. & Dzierzak, E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development 137, 3651–3661 (2010).

    Article  CAS  Google Scholar 

  8. Ng, C.E. et al. A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells. Stem Cells 28, 1869–1881 (2010).

    Article  CAS  Google Scholar 

  9. Yokomizo, T., Ng, C.E., Osato, M. & Dzierzak, E. Three-dimensional imaging of whole midgestation murine embryos show an intravascular localization for all hematopoietic clusters. Blood 117, 6132–6134 (2011).

    Article  CAS  Google Scholar 

  10. Garcia-Porrero, J.A., Godin, I.E. & Dieterlen-Lievre, F. Potential intraembryonic hemogenic sites at pre-liver stages in the mouse. Anat. Embryol. (Berl.) 192, 425–435 (1995).

    Article  CAS  Google Scholar 

  11. Garcia-Porrero, J.A. et al. Antigenic profiles of endothelial and hemopoietic lineages in murine intraembryonic hemogenic sites. Dev. Comp. Immunol. 22, 303–319 (1998).

    Article  CAS  Google Scholar 

  12. Ghiaur, G. et al. Rac1 is essential for intraembryonic hematopoiesis and for the initial seeding of fetal liver with definitive hematopoietic progenitor cells. Blood 111, 3313–3321 (2008).

    Article  CAS  Google Scholar 

  13. Jaffredo, T. et al. Aortic remodelling during hemogenesis: is the chicken paradigm unique? Int. J. Dev. Biol. 54, 1045–1054 (2010).

    Article  Google Scholar 

  14. Zovein, A.C. et al. Vascular remodeling of the vitelline artery initiates extravascular emergence of hematopoietic clusters. Blood 116, 3435–3444 (2010).

    Article  CAS  Google Scholar 

  15. Ferkowicz, M.J. & Yoder, M.C. (eds). Whole Embryo Imaging of Hematopoietic Cell Emergence and Migration 143–155 (Springer Science-Business Media, 2008).

  16. Yokomizo, T. et al. Requirement of Runx1/AML1/PEBP2αB for the generation of haematopoietic cells from endothelial cells. Genes Cells 6, 13–23 (2001).

    Article  CAS  Google Scholar 

  17. Yoshida, H. et al. Hematopoietic tissues, as a playground of receptor tyrosine kinases of the PDGF-receptor family. Dev. Comp. Immunol. 22, 321–332 (1998).

    Article  CAS  Google Scholar 

  18. Hogan, B., Beddington, R., Constantini, F. & Lacy, E. Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press, 1994).

  19. Becker, K., Jahrling, N., Kramer, E.R., Schnorrer, F. & Dodt, H.U. Ultramicroscopy: 3D reconstruction of large microscopical specimens. J. Biophotonics 1, 36–42 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the members of the laboratory for critical comments related to this work and also thank the following funding organizations: the US National Institutes of Health grants R37DK054077 (E.D.) and RO1HL091724 (N.A.S.), the Netherlands BSIK Innovation Program 03040 (E.D.) and the European Science Foundation Program EuroSTELLS 01-011 (E.D.), and the Netherlands Genomics Initiative—Cancer Genomic Distinguished Scientist Award (N.A.S.).

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Authors and Affiliations

  1. Department of Cell Biology, Erasmus Medical Center Stem Cell Institute, Erasmus Medical Center, Rotterdam, The Netherlands

    Tomomasa Yokomizo, Tomoko Yamada-Inagawa & Elaine Dzierzak

  2. Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore

    Tomomasa Yokomizo

  3. Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Amanda D Yzaguirre, Michael J Chen & Nancy A Speck

Authors
  1. Tomomasa Yokomizo
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  2. Tomoko Yamada-Inagawa
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  3. Amanda D Yzaguirre
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  4. Michael J Chen
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  5. Nancy A Speck
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  6. Elaine Dzierzak
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Correspondence to Tomomasa Yokomizo.

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Supplementary information

Supplementary Video 1

3D reconstructed video of a whole E9 embryo (14 sp). Ly6A-GFP transgenic embryo was stained with anti-GFP antibody (green) and anti-CD31 antibody (magenta). Picture was taken by x10 objective (HC PL APO CS x10/NA 0.4). Video was generated by Bitplane Imaris (Bitplane AG, Zurich Switzerland) assembling 311 slices (optical sections with 1.3 µm per z-step). (MOV 6085 kb)

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Yokomizo, T., Yamada-Inagawa, T., Yzaguirre, A. et al. Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos. Nat Protoc 7, 421–431 (2012). https://doi.org/10.1038/nprot.2011.441

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