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The fetal mouse metatarsal bone explant as a model of angiogenesis

Nature Protocols volume 10, pages 1459–1473 (2015)Cite this article

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Abstract

The mouse fetal metatarsal provides a unique tool for studying angiogenesis. In comparison with other commonly used in vitro or ex vivo angiogenesis assays, vessel outgrowth from mouse fetal metatarsals is more representative of sprouting angiogensis in vivo. It allows the analysis of blood vessel growth, and the mechanisms underpinning this process, in a multicellular microenvironment that drives the formation of a robust and complex vascular network in the absence of exogenous growth factors. By labeling different constituents of the vascular structure, it is possible to perform 3D rendering of the spatial interplay between different cellular components and to carry out quantitative analysis of vessel outgrowth. High-resolution imaging permits the visualization of fine structural and cellular details. As the assay involves the use of fetal tissues, it is possible to follow new blood vessel formation in genetically modified mice that are perinatally lethal. The entire process takes 9–13 d. A detailed description of how to set up and perform the assay is described here.

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Figure 1: Blood vessel formation from aortic rings and metatarsals.
Figure 2
Figure 3: Optimization of the mouse fetal metatarsal assay.
Figure 4: Immunofluorescence staining of metatarsals.
Figure 5: Optimization of metatarsal dissection procedure.
Figure 7: Example of image rendering for quantification of vascular growth from metatarsals.
Figure 8: Example of image processing for total vessel area analysis by the TRI2 software.
Figure 6: Example of exclusion criteria.
Figure 9: Treatment of metatarsal with proangiogenic and antiangiogenic factors.
Figure 10: Gene and protein expression profile in metatarsal bones or cells migrating from metatarsals.

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Acknowledgements

We thank D.L. Becker and L.E. Madden for their expertise in confocal microscopy. We thank S. Chai for his help with the Imaris software. This work was sponsored by grants from the Singapore Ministry of Education Academic Research Fund Tier 1 (2013-T1-002-051) and the ingapore Ministry of Education Academic Research Fund Tier 2 (MOE2014-T2-1-036) to X.W., by a National Medical Research Council—Cooperative Basic Research Grant (CBRG13nov094) to X.W. and W.H., and by a Medical Research Council UK grant (002973) awarded to J.G., S.E.M. and X.W.

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Author notes

  1. Weihua Song and Chee Wai Fhu: These authors contributed equally to this work.

Authors and Affiliations

  1. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

    Weihua Song, Chee Wai Fhu, Cheng Hao Liu, Nurul Azizah Binte Johari, Daniel Lio & Xiaomeng Wang

  2. Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Proteos, Singapore, Singapore

    Koon Hwee Ang, Wanjin Hong & Xiaomeng Wang

  3. Institute of Cardiovascular Sciences, Manchester, UK

    Sabu Abraham

  4. Department of Cell Biology, Institute of Ophthalmology, University College London, London, UK

    Stephen E Moss, John Greenwood & Xiaomeng Wang

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  1. Weihua Song
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Contributions

H.S. and X.W. optimized the protocol and designed the experiment. W.S., C.W.F., K.H.A., C.H.L., N.A.B.J., D.L., S.A. and X.W. performed the experiment. All authors contributed to the figure and manuscript preparation.

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Correspondence to Xiaomeng Wang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparison of vessel outgrowth between metatarsals and metacarpals.

Vessel outgrowths are labeled by PECAM-1 7 days after seeding. The total vascular area is normalized by bone size. Metacarpals lead to more vessel outgrowths compared to metatarsals. n=3 independent experiments, 6 bones are analyzed per group, data are mean±s.e.m. **P<0.01 (Student’s t-test). Scale bar, 320μm. Institutional ethical regulatory board permission was obtained at Nanyang Technological University.

Supplementary Figure 2 Comparison of the number of proliferating cells in the metatarsal microvasculature over time.

Proliferating cells are labelled by Ki67 antibody and blood vessels visualized by PECAM1 staining. The number of proliferating cells/50µm of blood vessels reaches the highest level 5 days after seeding and decrease dramatically from 8 days after seeding. n=3 independent experiments, 6 metatarsals per time point, data are mean±s.e.m. *P<0.05 (Student’s t-test). Scale bar, 10μm. Institutional ethical regulatory board permission was obtained at Nanyang Technological University.

Supplementary Figure 3 Visualization of vessel outgrowth and fibroblast migration from the metatarsals by phase contrast microscopy.

Fibroblasts migrate out from metatarsals on day 2 and blood vessels are visible by phase contrast microscopy from day 4 onwards (red arrow). Scale bar, 250μm. Institutional ethical regulatory board permission was obtained at Nanyang Technological University.

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Song, W., Fhu, C., Ang, K. et al. The fetal mouse metatarsal bone explant as a model of angiogenesis. Nat Protoc 10, 1459–1473 (2015). https://doi.org/10.1038/nprot.2015.097

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